Modified annexin compositions and methods of using same

ABSTRACT

Modified annexin proteins, including a homodimer of human annexin V, are provided. Methods for their use, such as to prevent thrombosis without increasing hemorrhage and to attenuate ischemia-reperfusion injury (IPI), are also provided. The modified annexins bind phosphatidylserine (PS) on cell surfaces, thereby preventing the assembly of the prothromkinase complex. The modified annexin decreases the binding of leukocytes and platelets during post-ischemic reperfusion, thereby restoring microvascular blood flow and decreasing organ damage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/267,837, “Modified Annexin Proteins and Methods for Their Use inOrgan Transplantation,” filed Nov. 3, 2005, which is a continuation inpart of U.S. application Ser. No. 11/078,231, “Modified Annexin Proteinsand Methods for Preventing Thrombosis,” filed Mar. 10, 2005, which is acontinuation in part of U.S. application Ser. No. 10/080,370, “ModifiedAnnexin Proteins and Methods for Preventing Thrombosis,” now U.S. Pat.No. 6,962,903, filed Feb. 21, 2002, which claims the benefit under 35U.S.C. §119 of U.S. Provisional Application No. 60/270,402, “Optimizingthe Annexin Molecule for Preventing Thrombosis,” filed Feb. 21, 2001,and U.S. Provisional Application No. 60/332,582, “Modified AnnexinMolecule for Preventing Thrombosis and Reperfusion Injury,” filed Nov.21, 2001. U.S. application Ser. No. 11/078,231 also claims the benefit,under 35 U.S.C. §119 of U.S. Provisional application No. 60/552,428,“The Use Of Modified Annexin To Attenuate Reperfusion Injury,” filedMar. 11, 2004, and U.S. Provisional application No. 60/579,589 “Use of aModified Annexin to Attenuate Reperfusion Injury,” filed Jun. 14, 2004.The disclosure of each of the foregoing patent applications is herebyincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions fortreating ischemia-reperfusion injury in organ transplantation and inother medical and surgical disorders. More particularly, it relates tomodified annexin proteins and methods for their use.

BACKGROUND OF THE INVENTION

Thrombosis—the formation, development, or presence of a blood clot(thrombus) in a blood vessel—is the most common severe medical disorder.The most frequent example of arterial thrombosis is coronary thrombosis,which leads to occlusion of the coronary arteries and often tomyocardial infarction (heart attack). More than 1.3 million patients areadmitted to the hospital for myocardial infarction each year in NorthAmerica. The standard therapy is administration of a thrombolyticprotein by infusion. Thrombolytic treatment of acute myocardialinfarction is estimated to save 30 lives per 1000 patients treated;nevertheless the 30-day mortality for this disorder remains substantial(Mehta et al., Lancet 35 6:449-454 (2000)). The disclosure of Mehta, etal., and the disclosure of all other patents, patent applications andpublications referred to herein, are incorporated herein by reference intheir entirety). It would be convenient to administer antithrombotic andthrombolytic agents by bolus injection, since they might be used beforeadmission to hospital with additional benefit (Rawles, J. Am. Coll.Cardiol. 30:1181-1186 (1997), incorporated herein by reference).However, bolus injection (as opposed to a more gradual intravenousinfusion) significantly increases the risk of cerebral hemorrhage (Mehtaet al., 2000). The development of an agent able to prevent thrombosisand/or increase thrombolysis, without augmenting the risk of bleeding,would be desirable.

Unstable angina, caused by inadequate oxygen delivery to the heart dueto coronary occlusion, is the most common cause of admission tohospital, with 1.5 million cases a year in the United States alone. Whenpatients with occlusion of coronary arteries are treated withangioplasty and stenting, the use of an antibody against platelet gpIIb/IIIa decreases the likelihood of restenosis. However, the sameantibody has shown no benefit in unstable angina without angioplasty,and a better method for preventing coronary occlusion in these patientsis needed.

Another important example of arterial thrombosis is cerebral thrombosis.Intravenous recombinant tissue plasminogen activator (rtPA) is the onlytreatment for acute ischemic stroke that is approved by the Food andDrug Administration. The earlier it is administered the better (Ernst etal., Stroke 31:2552-2557 (2000), incorporated herein by reference).However, intravenous rtPA administration is associated with increasedrisk of intracerebral hemorrhage. Full-blown strokes are often precededby transient ischemic attacks (TIA), and it is estimated that about300,000 persons suffer TIA every year in the United States. It would bedesirable to have a safe and effective agent that could be administeredas a bolus and would for several days prevent recurrence of cerebralthrombosis without increasing the risk of cerebral hemorrhage.Thrombosis also contributes to peripheral arterial occlusion indiabetics and other patients, and an efficacious and safe antithromboticagent for use in such patients is needed.

Venous thrombosis is a frequent complication of surgical procedures suchas hip and knee arthroplasties. It would be desirable to preventthrombosis without increasing hemorrhage into the field of operation.Similar considerations apply to venous thrombosis associated withpregnancy and parturition. Some persons are prone to repeated venousthrombotic events and are currently treated by antithrombotic agentssuch as coumarin-type drugs. The dose of such drugs must be titrated ineach patient, and the margin between effective antithrombotic doses andthose increasing hemorrhage is small. Having a treatment with betterseparation of antithrombotic activity from increased risk of bleeding isdesirable. All of the recently introduced antithrombotic therapies,including ligands of platelet gp IIb/IIIa, low molecular weightheparins, and a pentasaccharide inhibitor of factor Xa, carry anincreased risk of bleeding (Levine et al., Chest 119:108S-121S (2001),incorporated herein by reference). Hence there is a need to explorealternative strategies for preventing arterial and venous thrombosiswithout augmenting the risk of hemorrhage.

To inhibit the extension of arterial or venous thrombi withoutincreasing hemorrhage, it is necessary to exploit potential differencesbetween mechanisms involved in hemostasis and those involved inthrombosis in large blood vessels. Primary hemostatic mechanisms includethe formation of platelet microaggregates, which plug capillaries andaccumulate over damaged or activated endothelial cells in small bloodvessels. Inhibitors of platelet aggregation, including agentssuppressing the formation or action of thromboxane A₂, ligands of gpIIa/IIIb, and drugs acting on ADP receptors such as clopidogrel(Hallopeter, Nature 409:202-207 (2001), incorporated herein byreference), interfere with this process and therefore increase the riskof bleeding (Levine et al., 2001). In contrast to microaggregateformation, occlusion by an arterial or venous thrombus requires thecontinued recruitment and incorporation of platelets into the thrombus.To overcome detachment by shear forces in large blood vessels, plateletsmust be bound tightly to one another and to the fibrin network depositedaround them.

Evidence has accumulated that the formation of tight macroaggregates ofplatelets is facilitated by a cellular and a humoral amplificationmechanism, which reinforce each other. In the cellular mechanism, theformation of relatively loose microaggregates of platelets, induced bymoderate concentrations of agonists such as ADP, thromboxane A₂, orcollagen, is accompanied by the release from platelet α-granules of the85-kD protein Gas6 (Angelillo-Scherrer et al., Nature Medicine 7:215-221(2001), incorporated herein by reference). Binding of released Gas6 toreceptor tyrosine kinases (Axl, Sky, Mer) expressed on the surface ofplatelets induces complete degranulation and the formation of tightmacroaggregates of these cells. In the humoral amplification mechanism,a prothrombinase complex is formed on the surface of activated plateletsand microvesicles. This generates thrombin and fibrin. Thrombin isitself a potent platelet activator and inducer of the release of Gas6(Ishimoto and Nakano, FEBS Lett. 446:197-199 (2000), incorporated hereinby reference). Fully activated platelets bind tightly to the fibrinnetwork deposited around them. Histological observations show that bothplatelets and fibrin are necessary for the formation of a stablecoronary thrombus in humans (Falk et al. Interrelationship betweenatherosclerosis and thrombosis. In Vanstraete et al. (editors),Cardiovascular Thrombosis: Thrombocardiology and Thromboneurology.Philadelphia: Lipincott-Raven Publishers (1998), pp. 45-58, incorporatedherein by reference). Another platelet adhesion molecule, amphoterin, istranslocated to the platelet surface during activation, and bindsanionic phospholipid (Rouhainen et al., Thromb. Hemost. 84:1087-1094(2000), incorporated herein by reference). Like Gas6, amphoterin couldform a bridge during platelet aggregation.

The question arises whether it is possible to inhibit theseamplification mechanisms but not the initial platelet aggregation step,thereby preventing thrombosis without increasing hemorrhage. Theimportance of cellular amplification has recently been established bystudies of mice with targeted inactivation of Gas6 (Angelillo-Scherreret al., 2001). The Gas6−/−mice were found to be protected againstthrombosis and embolism induced by collagen and epinephrine. However,the Gas6−/−mice did not suffer from spontaneous hemorrhage and hadnormal bleeding after tail clipping. Furthermore, antibodies againstGas6 inhibited platelet aggregation in vitro as well as thrombosisinduced in vivo by collagen and epinephrine. In principle, suchantibodies, or ligands competing for Gas6 binding to receptor tyrosinekinases, might be used to inhibit thrombosis. However, in view of thepotency of humoral amplification, it might be preferable to inhibit thatstep. Ideally such an inhibitor would also have additional suppressiveactivity on the Gas6-mediated cellular amplification mechanism.

A strategy for preventing both cellular and humoral amplification ofplatelet aggregation is provided by the annexins, a family of highlyhomologous antithrombotic proteins of which ten are expressed in severalhuman tissues (Benz and Hofmann, Biol. Chem. 378:177-183 (1997),incorporated herein be reference). Annexins share the property ofbinding calcium and negatively charged phospholipids, both of which arerequired for blood coagulation. Under physiological conditions,negatively charged phospholipid is mainly supplied by phosphatidylserine(PS) in activated or damaged cell membranes. In intact cells, PS isconfined to the inner leaflet of the plasma membrane bilayer and is notaccessible on the surface. When platelets are activated, the amounts ofPS accessible on their surface, and therefore the extent of annexinbinding, are greatly increased (Sun et al., Thrombosis Res. 69:289-296(1993), incorporated herein by reference). During activation ofplatelets, microvesicles are released from their surfaces, greatlyincreasing the surface area expressing anionic phospholipids withprocoagulant activity (Merten et al., Circulation 99:2577-2582 (1999);Chow et al., J. Lab. Clin. Med. 135:66-72 (2000), both incorporatedherein by reference). These may play an important role in thepropagation of platelet-mediated arterial thrombi.

Proteins involved in the blood coagulation cascade (factors X, Xa, andVa) bind to membranes bearing PS on their surfaces, and to one another,forming a stable, tightly bound prothrombinase complex. Severalannexins, including I, II, IV, V, and VIII, bind PS with high affinity,thereby preventing the formation of a prothrombinase complex andexerting antithrombotic activity. Annexin V binds PS with very highaffinity (K_(d)=1.7 nmol/L), greater than the affinity of factors X, Xa,and Va for negatively charged phospholipids (Thiagarajan and Tait, J.Biol. Chem. 265:17420-17423 (1990), incorporated herein by reference).Tissue factor-dependent blood coagulation on the surface of activated ordamaged endothelial cells also requires surface expression of PS, andannexin V can inhibit this process (van Heerde et al., Arterioscl.Thromb. 14:824-830 (1994), incorporated herein by reference), althoughannexin is less effective in this activity than in inhibition ofprothrombinase generation (Rao et al., Thromb. Res. 62:517-531 (1992),incorporated herein by reference).

The binding of annexin V to activated platelets and to damaged cellsprobably explains the selective retention of the protein in thrombi.This has been shown in experimental animal models of venous and arterialthrombosis (Stratton et al., Circulation 92:3113-3121 (1995);Thiagarajan and Benedict, Circulation 96:2339-2347 (1997), bothincorporated herein by reference), and labeled annexin has been proposedfor medical imaging of vascular thrombi in humans, with reduced noiseand increased safety (Reno and Kasina, International Patent ApplicationPCT/US95/07599 (WO 95/34315) (published Dec. 21, 1995), incorporatedherein by reference). The binding to thrombi of a potent antithromboticagent such as annexin V provides a strategy for preventing the extensionor recurrence of thrombosis. Transient myocardial ischemia alsoincreases annexin V binding (Dumont et al., Circulation 102:1564-1568(2000), incorporated herein by reference). Annexin V imaging in humanshas shown increased binding of the protein in transplanted hearts whenendomyocardial biopsy has demonstrated vascular rejection (Acio et al.,J. Nuclear Med. 41 (5 Suppl.):127P (2000), incorporated herein byreference). This binding is presumably due to PS exteriorized on thesurface of damaged endothelial cells, as well as of apoptotic myocytesin hearts that are being rejected. It follows that administration ofannexin after myocardial infarction should prevent the formation ofpro-thrombotic complexes on both platelets and endothelial cells,thereby preventing the extension or recurrence of thrombosis. Annexin Vbinding is also augmented following cerebral hypoxia in humans(D'Arceuil et al., Stroke 2000: 2692-2700 (2000), incorporated herein byreference), which supports the hypothesis that administration of annexinfollowing TIA may decrease the likelihood of developing a full-blownstroke.

Annexins have shown anticoagulant activity in several in vitrothrombin-dependent assays, as well as in experimental animal models ofvenous thrombosis (Römisch et al., Thrombosis Res. 61:93-104 (1991); VanRyn-McKenna et al., Thrombosis Hemostasis 69:227-230 (1993), bothincorporated herein by reference) and arterial thrombosis (Thiagarajanand Benedict, 1997). Remarkably, annexin in antithrombotic doses had nodemonstrable effect on traditional ex vivo clotting tests in treatedrabbits (Thiagarajan and Benedict, 1997) and did not significantlyprolong bleeding times of treated rats (Van Ryn-McKenna et al., 1993).In treated rabbits annexin did not increase bleeding into a surgicalincision (Thiagarajan and Benedict, 1997). Thus, uniquely among all theagents so far investigated, annexins exert antithrombotic activitywithout increasing hemorrhage. Annexins do not inhibit plateletaggregation triggered by collagen or thrombin (Sun, et al., ThrombosisRes. 69: 281, 1993)), and platelet aggregation is the primary hemostaticmechanism. In the walls of damaged blood vessels and in extravasculartissues, the tissue factor/VIIa complex also exerts hemostatic effects,and this system is less susceptible to inhibition by annexin V than isthe prothrombinase complex (Rao et al., 1992). This is one argument forconfining administered annexin V to the vascular compartment as far aspossible; the risk of hemorrhage is likely to be reduced.

Despite such promising results for preventing thrombosis, a majorproblem associated with the therapeutic use of annexins is their shorthalf-life in the circulation, estimated in experimental animals to be 5to 15 minutes (Römisch et al., 1991; Stratton et al., 1995; Thiagarajanand Benedict, 1997); annexin V also has a short half-life in thecirculation of humans (Strauss et al., J. Nuclear Med. 41 (5 Suppl.):149P (2000), incorporated herein by reference). Most of the annexin islost into the urine, as expected of a 36 kDa protein (Thiagarajan andBenedict, 1997). There is a need, therefore, for a method of preventingannexin loss from the vascular compartment into the extravascularcompartment and urine, thereby prolonging antithrombotic activityfollowing a single injection.

Organ transplantation is a widely used procedure in many countries. Itallows survival of patients who would otherwise die of heart, liver orlung disease, and provides a better quality of life for patients onrenal dialysis. Because there is a shortage of organs fortransplantation, it would be advantageous if organs from non-ideal,extended-criteria donors could be transplanted successfully.Pretransplant correlates of diminished graft survival include advanceddonor age, longstanding donor hypertension or diabetes mellitus,non-heartbeating cadaver donors and prolonged cold preservation time (A.O. Ojo et al. J. Am. Soc. Nephrol. 2001; 12: 589). The outcome of livertransplants is less successful if the donor organs are steatotic (Amersiet al. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 8915). Accumulation offat in the liver is common, especially among ageing donors.

Despite advances in surgical technique, patient management andimmunosuppression, ischemia-reperfusion injury (IRI) remains animportant clinical problem. During recovery and preservation organs areanoxic, as they are in ischemia, and following transplantation they arereperfused. This results in IRI, which is estimated to account for asmuch as 10% of early graft loss in the case of transplanted livers(Amersi et al. J. Clin. Invest. 1999; 104: 1631). In addition, ischemiaof longer than 12 hours is highly correlated with primary nonfunction oftransplanted livers, as well as an increase incidence of both acute andchronic rejection (Fellstrom et al. Transplant Proc. 1998; 30: 4278).

Despite many attempts, reviewed by Selzner et al. (Gastroenterology2003; 125: 917), no method for decreasing IRI has become widely used inorgan grafting. It would be desirable to develop a therapeutic agent orprocedure which attenuates IRI following organ transplantation.

SUMMARY OF THE INVENTION

The present invention provides compounds and methods for preventingarterial or venous thrombosis. Recombinant human annexins are modifiedin such a way that its half-life in the vascular compartment isprolonged. This can be achieved in a variety of ways; three embodimentsare an annexin coupled to polyethylene glycol, a homopolymer orheteropolymer of annexin, and a fusion protein of annexin with anotherprotein (e.g., the Fc portion of immunoglobulin). The modified annexinbinds with high affinity to phosphatidylserine on the surface ofactivated platelets or injured cells, thereby preventing the binding ofGas6 as well as procoagulant proteins and the formation of aprothrombinase complex. Modified annexin therefore inhibits both thecellular and humoral mechanisms by which platelet aggregation isamplified, thereby preventing thrombosis.

In one embodiment, the present invention provides an isolated modifiedannexin protein containing an annexin protein, preferably annexin V,coupled to polyethylene glycol (PEG). Preferably, at least two PEGchains are coupled to a single annexin molecule, with each PEG having amolecular weight of at least 5 kDa, more preferably at least 10 kDa, andmost preferably at least 15 kDa. In an alternative embodiment, anisolated modified annexin protein contains an annexin protein coupled toat least one additional protein, such as an additional annexin protein(forming a homodimer) or the Fc portion of immunoglobulin. Theadditional protein preferably has a molecular weight of at least 30 kDa.Also provided by the present invention are pharmaceutical compositionscontaining an antithrombotically effective amount of any of the modifiedannexin proteins of the invention.

In methods of the invention, the modified annexin is administered to asubject at risk of thrombosis in a pharmaceutical composition having anantithrombotically effective amount of any one of the modified annexinproteins of the present invention. For example, the pharmaceuticalcomposition can be administered after an arterial thrombosis such ascoronary thrombosis, cerebral thrombosis, or a transient cerebralischemic attack. It can also be administered after a surgical operationassociated with venous thrombosis. Additionally, it can be administeredto subjects having conditions subject to arterial or venous thrombosis,such as diabetes, pregnancy, or parturition.

Also provided by the present invention are an isolated nucleic acidmolecule encoding a homodimer of annexin, a recombinant moleculecontaining at least a portion of the nucleic acid molecule, and arecombinant cell containing at least a portion of the nucleic acidmolecule. The recombinant cell is cultured under suitable conditions ina method of the invention to produce a homodimer of annexin.

The present invention also provides a method for screening for amodified annexin protein that modulates thrombosis using a thrombosistest system. In some embodiments, the test system is an in vitrocoagulation assay (Kuypers et al., Blood 104, 441a, 2004). In thisembodiment, the assay comprises human cells with externalized PS towhich purified human blood coagulation proteins bind and generate aprothrombin complex. The modified annexin binds to PS, competing withthe coagulation proteins, and thereby inhibits thrombin generation.

Also provided by the present invention is a method for in vivo screeningfor a modified annexin protein. In this method, a thrombosis animalmodel is contacted with a test modified annexin protein, after which thein vivo anticoagulation activity and increase in hemorrhage of the testmodified annexin protein is assessed. The anticoagulation activity andtime are compared with the anticoagulation activity and time of annexin,and the amount of hemorrhage is compared with hemorrhage in the animalmodel in the absence of the test modified annexin protein.

Thus the invention provides a method of treating a subject at risk ofthrombosis comprising administering to said subject anantithrombotically effective amount of an isolated modified annexinprotein comprising an annexin dimer. The isolated modified annexinprotein is administered after coronary thrombosis, after an overtcerebral thrombosis, after transient cerebral ischemic attack, after asurgical operation associated with venous thrombosis, wherein saidsubject is diabetic and said thrombosis is arterial thrombosis, orduring a condition selected from the group consisting of pregnancy andparturition. The isolated modified annexin protein is administered in arange from 0.1 mg/kg to 1.0 mg/kg.

The present invention also provides a method of inhibiting theattachment of leukocytes to endothelial cells comprising administeringan effective amount of an isolated modified annexin protein comprisingan annexin dimer to a patient in need thereof. In some embodiments, themethod further comprises reducing endothelial cell damage.

The present invention also provides a method of treating a subject atrisk of thrombosis comprising administering to said subject anantithrombotically effective amount of a protein having an affinity forphosphatidylserine that is at least 10% of the affinity of annexin V forphosphatidylserine, including wherein said protein is a monoclonal orpolyclonal antibody.

The present invention provides a method of protecting organs or tissuesusceptible to IRI, comprising contacting said organs or tissue withisolated modified annexin protein comprising an annexin dimer. In someembodiments, said organs or tissue are contacted with said isolatedmodified annexin protein comprising an annexin dimer by administering toa patient who is a recipient of an organ transplant, a therapeuticcomposition comprising isolated modified annexin protein comprising anannexin dimer. In some embodiments, the annexin dimer is administered inan intravascular dose of 10 to 1000 μ/kg. In other embodiments, theannexin dimer is administered in an intravascular dose of 100 to 500μ/kg.

In some embodiments, the therapeutic composition is administered to therecipient patient up to six hours prior to the reperfusion period. Inother embodiments, the therapeutic composition is administered to therecipient patient up to one hour after the reperfusion period. In otherembodiments, the therapeutic composition is intravenously administeredto the recipient patient during the transplantation. In some embodimentsthe therapeutic composition is administered to a patient with coronary,cerebral or other thrombosis as soon as possible after thevaso-occlusive event. In another embodiment, the therapeutic compositionis administered during a surgical operation to decrease the likelihoodof thrombosis and/or embolism. In yet another embodiment the therapeuticcomposition is administered to an organ graft recipient before, duringor after the operation to attenuate ischemia-reperfusion injury.

In some embodiments, an isolated modified annexin protein comprising anannexin dimer comprises SEQ ID NO:6, SEQ ID NO:13, or SEQ ID NO:23.

In still other embodiments, the organs or tissue comprise organ ortissue transplants which are contacted with an isolated modified annexinprotein comprising an annexin dimer by perfusing or flushing them exvivo with a solution containing isolated modified annexin proteincomprising an annexin dimer in a concentration of about 0.1 to 1 mg/l.In some embodiments, wherein said organs or tissue are perfused with asolution containing, in addition to isolated modified annexin proteincomprising an annexin dimer, at least one component selected from thegroup consisting of electrolytes and cell-protecting agents.

The present invention also provides a method of protecting organ ortissue transplants against reperfusion injury and dysfunction,comprising contacting said organ or tissue with a preservation fluid forperfusion and storage or rinsing of the organ, or tissue transplantsprior to the implantation of said organ or tissue in a patient requiringsuch implantation, wherein said preservation fluid contains an isolatedmodified annexin protein comprising an annexin dimer in a concentrationsufficient to prevent IRI after ischemia.

In some embodiments, the isolated modified annexin protein comprising anannexin dimer is added in a concentration of about 0.1 to 1 mg/l ofpreservation or rinse fluid.

In other embodiments, the isolated modified annexin protein comprisingan annexin dimer comprises SEQ ID NO:6, SEQ ID NO:13, or SEQ ID NO:17.

The present invention also provides method of protecting organs againstIRI comprising administering an effective amount of an isolated modifiedannexin protein comprising an annexin dimer, individually or incombination to a patient undergoing surgical operation.

The present invention further provides a method of protecting organs ortissue susceptible to IRI, comprising contacting said organs or tissuewith an effective amount of a protein having an affinity forphosphatidylserine that is at least 10% of the affinity of annexin V forphosphatidylserine. In some embodiments, the protein is a monoclonal orpolyclonal antibody.

The present invention also provides an isolated polynucleotide selectedfrom the group consisting of:(a) an isolated polynucleotide comprising apolynucleotide sequence having at least 95% identity to thepolynucleotide sequence of SEQ ID NO:17 or SEQ ID NO:23; (b) an isolatedpolynucleotide comprising the polynucleotide of SEQ ID NO:17 or SEQ IDNO:23; (c) an isolated polynucleotide having at least 95% identity tothe polynucleotide of SEQ ID NO:17 or SEQ ID NO:23; (d) the isolatedpolynucleotide of SEQ ID NO:17 or SEQ ID NO:23; (e) an isolatedpolynucleotide comprising a polynucleotide sequence encoding apolypeptide sequence having at least 95% identity to the polypeptidesequence of SEQ ID NO:19 or SEQ ID NO:23; (f) an isolated polynucleotidecomprising a polynucleotide sequence encoding the polypeptide of SEQ IDNO:19 or SEQ ID NO:23; (g) an isolated polynucleotide having apolynucleotide sequence encoding a polypeptide sequence having at least95% identity to the polypeptide sequence of SEQ ID NO:19 or SEQ IDNO:23; (h) an isolated polynucleotide encoding the polypeptide of SEQ IDNO:19 or SEQ ID NO:23; (i) a polynucleotide which is the RNA equivalentof a polynucleotide of (a) to (h); and (j) a polynucleotide sequencecomplementary to a polynucleotide of (a) to (i), wherein thepolynucleotides of (a)-(k) encode a polypeptide having the ability tobind phosphatidylserine on cell surfaces.

The present invention also provides an isolated polypeptide selectedfrom the group consisting of: (a) an isolated polypeptide encoded by apolynucleotide comprising the sequence of SEQ ID NO:19 or SEQ ID NO:23;(b) an isolated polypeptide comprising a polypeptide sequence having atleast 95% identity to the polypeptide sequence of SEQ ID NO:19 or SEQ IDNO:23; (c) an isolated polypeptide comprising the polypeptide sequenceof SEQ ID NO:19 or SEQ ID NO:23; (d) an isolated polypeptide having atleast 95% identity to the polypeptide sequence of SEQ ID NO:19 or SEQ IDNO:23; and (e) the polypeptide sequence of SEQ ID NO:19 or SEQ ID NO:23;wherein the polypeptides of (a)-(e) bind phosphatidylserine on cellsurfaces.

In still further embodiments, the present invention provides a method ofincreasing the duration of survival of blood platelets, comprisingadding an isolated modified annexin protein comprising an annexin dimerto stored platelets. The addition may be in a platelet storage medium.The addition may also be in a patient to whom platelets wereadministered, including the case where the patient is the recipient of aliver graft, including a thrombocytopenic liver graft patient.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show the structural scheme of two modified annexinembodiments. FIG. 1A shows the structural scheme of human annexin Vhomodimer with a His-tag; FIG. 1B shows the structural scheme of thehuman annexin V homodimer without a His-tag. FIG. 1C shows a DNAconstruct for making a homodimer of annexin V.

FIGS. 2A-D show the results of flowcytometric analysis of a mixture ofnormal (1×10⁷/ml) and PS exposing (1×10⁷/ml) RBCs incubated with 0.2μg/ml biotinylated AV (FIG. 2A); 0.2 μg/ml biotinylated DAV (FIG. 2B);0.2 μg/ml biotinylated AV and 0.2 μg/ml nonbiotinylated DAV (FIG. 2C);and 0.2 μg/ml biotinylated DAV and 0.2 μg/ml nonbiotinylated AV (FIG.2D), in each case, followed by R-phycoerythrein-conjugated streptavidin.

FIGS. 3A-E illustrate the levels of AV or DAV in mouse circulation atvarious times after injection. FIGS. 3A-B show serum samples recovered 5minutes and 20 minutes after injection of AV into mice, respectively.FIGS. 3C-E show serum samples recovered 5 minutes, 25 minutes and 120minutes after injection of annexin V homodimer (DAV) into mice,respectively.

FIGS. 4A-C show PLA₂ -induced hemolysis of PS-exposing RBC. A mixture ofnormal (1×10⁷/ml) and PS exposing (1×10⁷/ml) RBCs was incubated with 100ng/ml pancreatic PLA₂ (pPLA₂ ) or secretory PLA₂ (sPLA2 ). Hemolysis wasmeasured as a function of time and expressed relative to 100% hemolysisinduced by osmotic shock. The percentage of PS-exposing cells wasdetermined by flow cytometry of the cell suspension after labeling withbiotinylated DAV and R-phycoerythrein-conjugated streptavidin. FIG. 4Ashows hemolysis induced by 100 ng/ml pPLA₂ in absence (triangles) orpresence of 2 μg/ml DAV (circles) or AV (squares). FIG. 4B showshemolysis induced by 100 ng/ml pPLA₂ in the presence of various amountsof DAV (circles) or AV (squares). FIG. 4C shows PS-exposing cells in thecell suspension after 60 minutes incubation with 100 ng/ml pPLA₂ in thepresence of 2 μg/ml DAV.

FIG. 5 shows serum alanine aminotransferase (ALT) levels in mice shamoperated (Sham), mice given saline, mice given HEPES buffer 6 hrs.before clamping the hepatic artery, mice given pegylated annexin (PEGAnex) or annexin dimer 6 hrs. before clamping the artery, and mice givenmonomeric annexin (Anex). The asterisk above PEG ANNEX and ANNEX DIMERindicates p<0.001.

FIG. 6 is a plot of clotting time of an in vitro clotting assaycomparing the anticoagulant potency of recombinant human annexin V andpegylated recombinant human annexin V.

FIG. 7 shows thrombus weight in the five treatment groups of the10-minute thrombosis study (mean±sd; n=8).

FIG. 8 shows APTT in the five treatment groups of the thrombosis study(mean±sd; n=8).

FIG. 9 shows bleeding time in the three groups of the tail bleedingstudy (mean±sd; n=8).

FIG. 10 shows blood loss in the three groups of the tail bleeding study(mean±sem; n=8).

FIG. 11 shows APTT in the three groups of the tail bleeding study(mean±sd; n=8).

FIG. 12A shows attachment of leukocytes to endothelial cells duringischemia-reperfusion injury with and without diannexin for periportalsinusoids. FIG. 12B shows attachment of leukocytes to endothelial cellsduring ischemia-reperfusion injury with and without diannexin forcentrilobular sinusoids.

FIG. 13A shows attachment of platelets to endothelial cells duringischemia-reperfusion injury with and without diannexin for periportalsinusoids. FIG. 13B shows attachment of platelets to endothelial cellsduring ischemia-reperfusion injury with and without diannexin forcentrilobular sinusoids.

FIG. 14A shows swelling of endothelial cells during ischemia-reperfusioninjury with and without diannexin for periportal sinusoids. FIG. 14Bshows swelling of endothelial cells during ischemia-reperfusion injurywith and without diannexin for centrilobular sinusoids.

FIG. 15A shows phagocytic activity of Kupffer cells duringischemia-reperfusion injury with and without diannexin for periportalsinusoids. FIG. 15B shows phagocytic activity of Kupffer cells duringischemia-reperfusion injury with and without diannexin for centrilobularsinusoids.

FIG. 16 shows protection by diannexin in ischemia-reperfusion injury insteatotic mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds and methods for preventingthrombosis in mammals without increasing hemorrhage. The inventionrelies in part on the recognition that the primary mechanisms ofplatelet aggregation are different from the mechanisms of amplifyingplatelet aggregation, which are required for the formation of anarterial or venous thrombus. By inhibiting thrombus formation but notprimary platelet aggregation, thrombosis can be prevented withoutincreasing hemorrhage.

Compounds of the invention include any product containing annexin aminoacid sequences that have been modified to increase the half-life of theproduct in humans or other mammals. Where “amino acid sequence” isrecited herein to refer to an amino acid sequence of anaturally-occurring protein molecule, “amino acid sequence” and liketerms, such as “polypeptide” or “protein,” are not meant to limit theamino acid sequence to the complete, native amino acid sequenceassociated with the recited proteins. The annexins are a family ofhomologous phospholipid-binding membrane proteins, of which tenrepresent distinct gene products expressed in mammals (Benz and Hofmann,1997). Crystallographic analysis has revealed a common tertiarystructure for all the family members so far studied, exemplified byannexin V (Huber et al., EMBO Journal 9:3867 (1990), incorporated hereinby reference). The core domain is a concave discoid structure that canbe closely apposed to phospholipid membranes. It contains foursubdomains, each consisting of a 70-amino-acid annexin repeat made up offive α-helices. The annexins also have a more hydrophilic tail domainthat varies in length and amino acid sequence among the differentannexins. The sequences of genes encoding annexins are well known (e.g.,Funakoshi et al., Biochemistry 26:8087-8092 (1987) (annexin V),incorporated herein by reference).

Annexin proteins include proteins of the annexin family, such as AnnexinII (lipocortin 2, calpactin 1, protein I, p36, chromobindin 8), AnnexinIII (lipocortin 3, PAP-III), Annexin IV (lipocortin 4, endonexin I,protein II, chromobindin 4), Annexin V (Lipocortin 5, Endonexin 2,VAC-alpha, Anchorin CII, PAP-I), Annexin VI (Lipocortin 6, Protein III,Chromobindin 20, p68, p70), Annexin VII (Synexin), Annexin VIII(VAC-beta), Annexin XI (CAP-50), and Annexin XIII (ISA).

Annexin IV shares many of the same properties of Annexin V. Like annexinV, Annexin IV binds to acidic phospholipid membranes in the presence ofcalcium. Annexin IV is a close structural homologue of Annexin V. Thesequence of Annexin IV is known. Hamman et al., Biochem. Biophys. Res.Comm., 156:660-667 (1988). Annexin IV belongs to the annexin family ofcalcium-dependent phospholipid binding proteins. Its functions are stillnot clearly defined.

Annexin IV (endonexin) is a 32kDa, calcium-dependent membrane-bindingprotein. The translated amino acid sequence of Annexin IV shows the fourdomain structure characteristic of proteins in this class. Annexin IVhas 45-59% identity with other members of its family and shares asimilar size and exon-intron organization. Isolated from human placenta,Annexin IV encodes a protein that has in vitro anticoagulant activityand inhibits phospholipase A₂ activity. Annexin IV is almost exclusivelyexpressed in epithelial cells.

Annexin VIII belongs to the family of Ca²⁺dependent phospholipid bindingproteins (annexins) and has high sequence identity to Annexin V (56%).Hauptmann, et al., Eur J Biochem. 1989 Oct. 20; 185(1):63-71. It wasinitially isolated as a 2.2 kb vascular anticoagulant-beta. Annexin VIIIis neither an extracellular protein nor associated with the cellsurface. It may not play a role in blood coagulation in vivo. Itsphysiological role remains unknown. It is expressed at low levels inhuman placenta and shows restricted expression in lung, endothelia andskin, liver and kidney.

In the present invention, annexin proteins are modified to increasetheir half-life in humans or other mammals. In some embodiments, theannexin protein is annexin V, annexin IV or annexin VIII. One suitablemodification of annexin is an increase in its effective size, whichprevents loss from the vascular compartment into the extravascularcompartment and urine, thereby prolonging antithrombotic activityfollowing a single injection. Any increase in effective size thatmaintains a sufficient binding affinity with phosphatidylserine iswithin the scope of the present invention.

In one embodiment of the invention, a modified annexin contains arecombinant human annexin protein coupled to polyethylene glycol (PEG)in such a way that the modified annexin is capable of performing thefunction of annexin in a phosphatidylserine (PS)-binding assay. Theantithrombotic action of the intravenously administered annexin-PEGconjugate is prolonged as compared with that of the free annexin. Therecombinant annexin protein coupled to PEG can be annexin V protein oranother annexin protein. In one embodiment, the annexin protein isannexin V, annexin IV or annexin VIII.

PEG consists of repeating units of ethylene oxide that terminate inhydroxyl groups on either end of a linear or, in some cases, branchedchain. The size and molecular weight of the coupled PEG chain dependupon the number of ethylene oxide units it contains, which can beselected. For the present invention, any size of PEG and number of PEGchains per annexin molecule can be used such that the half-life of themodified annexin is increased, relative to annexin, while preserving thefunction of binding of the modified molecule to PS. As stated above,sufficient binding includes binding that is diminished from that of theunmodified annexin, but still competitive with the binding of Gas6 andfactors of the prothrombinase complex and therefore able to preventthrombosis. The optimal molecular weight of the conjugated PEG varieswith the number of PEG chains. In one embodiment, two PEG molecules ofmolecular weight of at least about 15 kDa each are coupled to eachannexin molecule. The PEG molecules can be linear or branched. Thecalcium-dependent binding of annexins to PS is affected not only by thesize of the coupled PEG molecules, but also the sites on the protein towhich PEG is bound. Optimal selection ensures that desirable propertiesare retained. Selection of PEG attachment sites is facilitated byknowledge of the three-dimensional structure of the molecule and bymutational and crystallographic analyses of the interaction of themolecule with phospholipid membranes (Campos et al., Biochemistry37:8004-8008 (1998), incorporated herein by reference).

In the area of drug delivery, PEG derivatives have been widely used incovalent attachment (referred to as pegylation) to proteins to enhancesolubility, as well as to reduce immunogenicity, proteolysis, and kidneyclearance. The superior clinical efficacy of recombinant productscoupled to PEG is well established. For example, PEG-interferon alpha-2aadministered once weekly is significantly more effective againsthepatitis C virus than three weekly doses of the free interferon(Heathcote et al., N. Engl. J. Med. 343:1673-1680 (2000), incorporatedherein by reference). Coupling to PEG has been used to prolong thehalf-life of recombinant proteins in vivo (Knauf et al., J. Biol. Chem.266:2796-2804 (1988), incorporated herein by reference), as well as toprevent the enzymatic degradation of recombinant proteins and todecrease the immunogenicity sometimes observed with homologous products(references in Hermanson, Bioconjugate techniques. New York, AcademicPress (1996), pp. 173-176, incorporated herein by reference).

In another embodiment of the invention, the modified annexin protein isa polymer of annexin proteins that has an increased effective size. Itis believed that the increase in effective size results in prolongedhalf-life in the vascular compartment and prolonged antithromboticactivity. One such modified annexin is a dimer of annexin proteins. Inone embodiment, the dimer of annexin is a homodimer of annexin V,annexin IV or annexin VIII. In another embodiment, the dimer of annexinis a heterodimer of annexin V and other annexin protein (e.g., annexinIV or annexin VIII), annexin IV and another annexin protein (e.g.,annexin V or annexin VIII) or annexin VIII and another annexin protein(e.g., annexin V or annexin IV). Another such polymer is theheterotetramer of annexin II with p 11, a member of the S100 family ofcalcium-binding proteins. The binding of an S100 protein to an annexinincreases the affinity of the annexin for Ca²⁺. The annexin homopolymeror heterotetramer can be produced by bioconjugate methods or recombinantmethods, and be administered by itself or in a PEG-conjugated form.

In some embodiments, the modified annexins have increased affinity forPS. As described in Example 4, a homodimer of human annexin V (DAV) wasprepared in using well-established methods of recombinant DNAtechnology. The annexin molecules of the homodimer are joined throughpeptide bonds to a flexible linker (FIG. 1). In some embodiments, theflexible linker contains a sequence of amino acids flanked by a glycineand a serine residue at either end to serve as swivels. The linkerpreferably comprises one or more such “swivels.” Preferably, the linkercomprises 2 swivels which may be separated by at least 2 amino acids,more particularly by at least 4 amino acids, more particularly by atleast 6 amino acids, more particularly by at least 8 amino acids, moreparticularly by at least 10 amino acids. Preferably, the overall lengthof the linker is 5-30 amino acids, 5-20 amino acids, 5-10 amino acids,10-15 amino acids, or 10-20 amino acids. The dimer can fold in such away that the convex surfaces of the monomer, 2 which bind Ca² +and PS,can both gain access to externalized PS. Flexible linkers are known inthe art, for example, (GGGGS; SEQ ID NO: 24)(n) (n=3-4), and helicallinkers, (EAAAK; SEQ ID NO: 25)(n) (n=2-5), described in Arai, et al.,Proteins. 2004 Dec. 1; 57(4):829-38. As described in Example II, theannexin V homodimer out-competes annexin monomer in binding to PS oncell surfaces (FIG. 2).

In another embodiment of the invention, recombinant annexin is expressedwith, or chemically coupled to, another protein such as the Fc portionof immunoglobulin. Such expression or coupling increases the effectivesize of the molecule, preventing the loss of annexin from the vascularcompartment and prolonging its anticoagulant action.

Preferably, a modified annexin protein of the invention is an isolatedmodified annexin protein. The modified annexin protein can containannexin II, annexin IV, annexin V, or annexin VIII. In some embodiments,the protein is modified human annexin. In some embodiments, the modifiedannexin contains recombinant human annexin. According to the presentinvention, an isolated or biologically pure protein is a protein thathas been removed from its natural environment. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which theprotein has been purified. An isolated modified annexin protein of thepresent invention can be obtained from its natural source, can beproduced using recombinant DNA technology, or can be produced bychemical synthesis. As used herein, an isolated modified annexin proteincan be a full-length modified protein or any homologue of such aprotein. It can also be (e.g., for a pegylated protein) a modifiedfull-length protein or a modified homologue of such a protein.

The minimal size of a protein homologue of the present invention is asize sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid with the complementary sequence of a nucleicacid molecule encoding the corresponding natural protein. As such, thesize of the nucleic acid molecule encoding such a protein homologue isdependent on nucleic acid composition and percent homology between thenucleic acid molecule and complementary sequence as well as uponhybridization conditions per se (e.g., temperature, salt concentration,and formamide concentration). The minimal size of such nucleic acidmolecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode a protein homologue ofthe present invention is from about 12 to about 18 nucleotides inlength. There is no limit on the maximal size of such a nucleic acidmolecule in that the nucleic acid molecule can include a portion of agene, an entire gene, or multiple genes or portions thereof. Similarly,the minimal size of an annexin protein homologue or a modified annexinprotein homologue of the present invention is from about 4 to about 6amino acids in length, with sizes depending on whether a full-length,multivalent (i.e., fusion protein having more than one domain, each ofwhich has a function) protein, or functional portions of such proteinsare desired. Annexin and modified annexin homologues of the presentinvention preferably have activity corresponding to the natural subunit,such as being able to perform the activity of the annexin protein inpreventing thrombus formation.

Annexin protein and modified annexin homologues can be the result ofnatural allelic variation or natural mutation. The protein homologues ofthe present invention can also be produced using techniques known in theart, including, but not limited to, direct modifications to the proteinor modifications to the gene encoding the protein using, for example,classic or recombinant DNA techniques to effect random or targetedmutagenesis.

Also included is a modified annexin protein containing an amino acidsequence that is at least about 75%, more preferably at least about 80%,more preferably at least about 85%, more preferably at least about 90%,more preferably at least about 95%, and most preferably at least about98% identical to amino acid sequence SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:12, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23 or a protein encoded byan allelic variant of a nucleic acid molecule encoding a proteincontaining any of these sequences. Also included is a modified annexinprotein comprising more than one of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:12, SEQ ID NO:15; SEQ ID NO:19, or SEQ ID NO:23; for example, aprotein comprising SEQ ID NO:3 and SEQ ID NO:12 and separated by alinker. Methods to determine percent identities between amino acidsequences and between nucleic acid sequences are known to those skilledin the art. Methods to determine percent identities between sequencesinclude computer programs such as the GCG ® Wisconsin package™(available from Accelrys Corporation), the DNAsis™ program (availablefrom Hitachi Software, San Bruno, Calif.), the Vector NTI Suite(available from Informax, Inc., North Bethesda, Md.), or the BLASTsoftware available on the NCBI website.

In one embodiment, a modified annexin protein includes an amino acidsequence of at least about 5 amino acids, preferably at least about 50amino acids, more preferably at least about 100 amino acids, morepreferably at least about 200 amino acids, more preferably at leastabout 250 amino acids, more preferably at least about 275 amino acids,more preferably at least about 300 amino acids, and most preferably atleast about 319 amino acids or the full-length annexin protein,whichever is shorter. In another embodiment, annexin proteins containfull-length proteins, i.e., proteins encoded by full-length codingregions, or post-translationally modified proteins thereof, such asmature proteins from which initiating methionine and/or signal sequencesor “pro” sequences have been removed.

A fragment of a modified annexin protein of the present inventionpreferably contains at least about 5 amino acids, more preferably atleast about 10 amino acids, more preferably at least about 15 aminoacids, more preferably at least about 20 amino acids, more preferably atleast about 25 amino acids, more preferably at least about 30 aminoacids, more preferably at least about 35 amino acids, more preferably atleast about 40 amino acids, more preferably at least about 45 aminoacids, more preferably at least about 50 amino acids, more preferably atleast about 55 amino acids, more preferably at least about 60 aminoacids, more preferably at least about 65 amino acids, more preferably atleast about 70 amino acids, more preferably at least about 75 aminoacids, more preferably at least about 80 amino acids, more preferably atleast about 85 amino acids, more preferably at least about 90 aminoacids, more preferably at least about 95 amino acids, and even morepreferably at least about 100 amino acids in length.

In one embodiment, an isolated modified annexin protein of the presentinvention contains a protein encoded by a nucleic acid molecule havingthe nucleic acid sequence SEQ ID NO:4, SEQ ID NO:17 or SEQ ID NO:21.Alternatively, the modified annexin protein contains a protein encodedby a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1or by an allelic variant of a nucleic acid molecule having one of thesesequences. Alternatively, the modified annexin protein contains morethan one protein sequence encoded by a nucleic acid molecule having thenucleic acid sequence SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:13 or by anallelic variant of a nucleic acid molecule having this sequence.

In one embodiment, an isolated modified annexin protein of the presentinvention contains a protein encoded by a nucleic acid molecule havingthe nucleic acid sequence SEQ ID NO:10 or by an allelic variant of anucleic acid molecule having this sequence. Alternatively, the modifiedannexin protein contains more than one protein sequence encoded by anucleic acid molecule having the nucleic acid sequence SEQ ID NO:10 orby an allelic variant of a nucleic acid molecule having this sequence(e.g., SEQ ID NO:12-linker-SEQ ID NO:12; SEQ ID NO:19).

In another embodiment, an isolated modified annexin protein of thepresent invention is a modified protein encoded by a nucleic acidmolecule having the nucleic acid sequence SEQ ID NO:13 or by an allelicvariant of a nucleic acid molecule having this sequence. Alternatively,the modified annexin protein contains more than one protein sequenceencoded by a nucleic acid molecule having the nucleic acid sequence SEQID NO:13 or by an allelic variant of a nucleic acid molecule having thissequence (e.g., SEQ ID NO: 15-linker-SEQ ID NO: 15; SEQ ID NO:23).

In another embodiment, an isolated modified annexin protein of thepresent invention is a modified protein which contains a protein encodedby a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1and a protein encoded by a nucleic acid molecule having the nucleic acidsequence SEQ ID NO:10, or by allelic variants of these nucleic acidmolecules (e.g., SEQ ID NO: 3-linker-SEQ ID NO:12 or SEQ IDNO:12-linker-SEQ ID NO:3).

In another embodiment, an isolated modified annexin protein of thepresent invention is a modified protein which contains a protein encodedby a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1and a protein encoded by a nucleic acid molecule having the nucleic acidsequence SEQ ID NO:13, or by allelic variants of these nucleic acidmolecules (e.g., SEQ ID NO:3-linker-SEQ ID NO:15 or SEQ IDNO:15-linker-SEQ ID NO:3).

In another embodiment, an isolated modified annexin protein of thepresent invention is a modified protein which contains a protein encodedby a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:10and a protein encoded by a nucleic acid molecule having the nucleic acidsequence SEQ ID NO:13, or by allelic variants of these nucleic acidmolecules (e.g., SEQ ID NO:12-linker-SEQ ID NO:15 or SEQ IDNO:15-linker-SEQ ID NO:12).

One embodiment of the present invention includes a non-native modifiedannexin protein encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with an annexin gene. As used herein,stringent hybridization conditions refer to standard hybridizationconditions under which nucleic acid molecules, includingoligonucleotides, are used to identify molecules having similar nucleicacid sequences. Such standard conditions are disclosed, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Labs Press (1989), incorporated herein by reference. Stringenthybridization conditions typically permit isolation of nucleic acidmolecules having at least about 70% nucleic acid sequence identity withthe nucleic acid molecule being used to probe in the hybridizationreaction. Formulae to calculate the appropriate hybridization and washconditions to achieve hybridization permitting 30% or less mismatch ofnucleotides are disclosed, for example, in Meinkoth et al., Anal.Biochem. 138:267-284 (1984), incorporated herein by reference. In someembodiments, hybridization conditions will permit isolation of nucleicacid molecules having at least about 80% nucleic acid sequence identitywith the nucleic acid molecule being used to probe. In otherembodiments, hybridization conditions will permit isolation of nucleicacid molecules having at least about 90% nucleic acid sequence identitywith the nucleic acid molecule being used to probe. In still otherembodiments, hybridization conditions will permit isolation of nucleicacid molecules having at least about 95% nucleic acid sequence identitywith the nucleic acid molecule being used to probe.

A modified annexin protein includes a protein encoded by a nucleic acidmolecule that is at least about 50 nucleotides and that hybridizes underconditions that preferably allow about 20% base pair mismatch, morepreferably under conditions that allow about 15% base pair mismatch,more preferably under conditions that allow about 10% base pairmismatch, more preferably under conditions that allow about 5% base pairmismatch, and even more preferably under conditions that allow about 2%base pair mismatch with a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:17, SEQ ID NO:21, or a complement of any of these nucleic acidmolecules.

As used herein, an annexin gene includes all nucleic acid sequencesrelated to a natural annexin gene such as regulatory regions thatcontrol production of the annexin protein encoded by that gene (such as,but not limited to, transcription, translation or post-translationcontrol regions) as well as the coding region itself. In one embodiment,an annexin gene includes the nucleic acid sequence SEQ ID NO:1. Inanother embodiment, an annexin gene includes the nucleic acid sequenceSEQ ID NO:10. In another embodiment, an annexin gene includes thenucleic acid sequence SEQ ID NO:13. In another embodiment, an annexingene includes the nucleic acid sequence SEQ ID NO:17. In anotherembodiment, an annexin gene includes the nucleic acid sequence SEQ IDNO:21. It should be noted that since nucleic acid sequencing technologyis not entirely error-free, SEQ ID NO:1 (as well as other sequencespresented herein), at best, represents an apparent nucleic acid sequenceof the nucleic acid molecule encoding an annexin protein of the presentinvention.

In another embodiment, an annexin gene can be an allelic variant thatincludes a similar but not identical sequence to SEQ ID NO:1. In anotherembodiment, an annexin gene can be an allelic variant that includes asimilar but not identical sequence to SEQ ID NO:10. In anotherembodiment, an annexin gene can be an allelic variant that includes asimilar but not identical sequence to SEQ ID NO:13. In anotherembodiment, an annexin gene can be an allelic variant that includes asimilar but not identical sequence to SEQ ID NO:17. In anotherembodiment, an annexin gene can be an allelic variant that includes asimilar but not identical sequence to SEQ ID NO:21. An allelic variantof an annexin gene including SEQ ID NO:1 is a gene that occurs atessentially the same locus (or loci) in the genome as the gene includingSEQ ID NO:1, but which, due to natural variations caused by, forexample, mutation or recombination, has a similar but not identicalsequence. Allelic variants typically encode proteins having similaractivity to that of the protein encoded by the gene to which they arebeing compared. Allelic variants can also comprise alterations in the 5′or 3′ untranslated regions of the gene (e.g., in regulatory controlregions). Allelic variants are well known to those skilled in the artand would be expected to be found within a given human since the genomeis diploid and/or among a population comprising two or more humans.

An isolated modified annexin protein of the present invention can beobtained from its natural source, can be produced using recombinant DNAtechnology, or can be produced by chemical synthesis. As used herein, anisolated modified annexin protein can contain a full-length protein orany homologue of such a protein. Examples of annexin and modifiedannexin homologues include annexin and modified annexin proteins inwhich amino acids have been deleted (e.g., a truncated version of theprotein, such as a peptide or by a protein splicing reaction when anintron has been removed or two exons are joined), inserted, inverted,substituted and/or derivatized (e.g., by glycosylation, phosphorylation,acetylation, methylation, myristylation, prenylation, palmitoylation,amidation and/or addition of glycerophosphatidyl inositol) such that thehomologue includes at least one epitope capable of eliciting an immuneresponse against an annexin protein. That is, when the homologue isadministered to an animal as an immunogen, using techniques known tothose skilled in the art, the animal will produce a humoral and/orcellular immune response against at least one epitope of an annexinprotein. Annexin and modified annexin homologues can also be selected bytheir ability to selectively bind to immune serum. Methods to measuresuch activities are disclosed herein. Annexin and modified annexinhomologues also include those proteins that are capable of performingthe function of native annexin in a functional assay; that is, arecapable of binding to phosphatidylserine or to activated platelets orexhibiting antithrombotic activity. Methods for such assays aredescribed in the Examples section and elsewhere herein.

A modified annexin protein of the present invention may be identified byits ability to perform the function of an annexin protein in afunctional assay. The phrase “capable of performing the function of thatin a functional assay” means that the protein or modified protein has atleast about 10% of the activity of the natural protein in the functionalassay. In other embodiments, it has at least about 20% of the activityof the natural protein in the functional assay. In other embodiments, ithas at least about 30% of the activity of the natural protein in thefunctional assay. In other embodiments, it has at least about 40% of theactivity of the natural protein in the functional assay. In otherembodiments, it has at least about 50% of the activity of the naturalprotein in the functional assay. In other embodiments, the protein ormodified protein has at least about 60% of the activity of the naturalprotein in the functional assay. In still other embodiments, the proteinor modified protein has at least about 70% of the activity of thenatural protein in the functional assay. In yet other embodiments, theprotein or modified protein has at least about 80% of the activity ofthe natural protein in the functional assay. In other embodiments, theprotein or modified protein has at least about 90% of the activity ofthe natural protein in the functional assay. Examples of functionalassays are described herein.

An isolated protein of the present invention can be produced in avariety of ways, including recovering such a protein from a bacteriumand producing such a protein recombinantly. One embodiment of thepresent invention is a method to produce an isolated modified annexinprotein of the present invention using recombinant DNA technology. Sucha method includes the steps of (a) culturing a recombinant cellcontaining a nucleic acid molecule encoding a modified annexin proteinof the present invention to produce the protein and (b) recovering theprotein therefrom. Details on producing recombinant cells and culturingthereof are presented below. The phrase “recovering the protein” referssimply to collecting the whole fermentation medium containing theprotein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques.

Isolated proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein in afunctional assay.

Modified Annexin Nucleic Acid Molecules or Genes

Another embodiment of the present invention is an isolated nucleic acidmolecule capable of hybridizing under stringent conditions with a geneencoding a modified annexin protein such as a homodimer of annexin V, ahomodimer of annexin IV, a homodimer of annexin VIII, a heterodimer ofannexin V and annexin VIII, a heterodimer of annexin V and annexin IV ora heterodimer of annexin IV and annexin VIII. Such a nucleic acidmolecule is also referred to herein as a modified annexin nucleic acidmolecule. Included is an isolated nucleic acid molecule that hybridizesunder stringent conditions with a modified annexin gene. Thecharacteristics of such genes are disclosed herein. In accordance withthe present invention, an isolated nucleic acid molecule is a nucleicacid molecule that has been removed from its natural milieu (i.e., thathas been subject to human manipulation). As such, “isolated” does notreflect the extent to which the nucleic acid molecule has been purified.An isolated nucleic acid molecule can include DNA, RNA, or derivativesof either DNA or RNA.

As stated above, a modified annexin gene includes all nucleic acidsequences related to a natural annexin gene, such as regulatory regionsthat control production of an annexin protein encoded by that gene (suchas, but not limited to, transcriptional, translational, orpost-translational control regions) as well as the coding region itself.A nucleic acid molecule of the present invention can be an isolatedmodified annexin nucleic acid molecule or a homologue thereof. A nucleicacid molecule of the present invention can include one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof. The minimal size of a modified annexin nucleicacid molecule of the present invention is the minimal size capable offorming a stable hybrid under stringent hybridization conditions with acorresponding natural gene. Annexin nucleic acid molecules can alsoinclude a nucleic acid molecule encoding a hybrid protein, a fusionprotein, a multivalent protein or a truncation fragment.

An isolated nucleic acid molecule of the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. As used herein, the phrase “at least a portion of” an entityrefers to an amount of the entity that is at least sufficient to havethe functional aspects of that entity. For example, at least a portionof a nucleic acid sequence, as used herein, is an amount of a nucleicacid sequence capable of forming a stable hybrid with the correspondinggene under stringent hybridization conditions.

An isolated nucleic acid molecule of the present invention can also beproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning, etc.) or chemical synthesis.Isolated modified annexin nucleic acid molecules include natural nucleicacid molecules and homologues thereof, including, but not limited to,natural allelic variants and modified nucleic acid molecules in whichnucleotides have been inserted, deleted, substituted, and/or inverted insuch a manner that such modifications do not substantially interferewith the ability of the nucleic acid molecule to encode an annexinprotein of the present invention or to form stable hybrids understringent conditions with natural nucleic acid molecule isolates.

A modified annexin nucleic acid molecule homologue can be produced usinga number of methods known to those skilled in the art (see, e.g.,Sambrook et al., 1989). For example, nucleic acid molecules can bemodified using a variety of techniques including, but not limited to,classic mutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures, and ligation ofmixture groups to “build” a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule homologues can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid (e.g., the ability of ahomologue to elicit an immune response against an annexin protein and/orto function in a clotting assay, or other functional assay), and/or byhybridization with isolated annexin-encoding nucleic acids understringent conditions.

An isolated modified annexin nucleic acid molecule of the presentinvention can include a nucleic acid sequence that encodes at least onemodified annexin protein of the present invention, examples of suchproteins being disclosed herein. Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a modified annexinprotein.

One embodiment of the present invention is a modified annexin nucleicacid molecule that is capable of hybridizing under stringent conditionsto a nucleic acid strand that encodes at least a portion of a modifiedannexin protein or a homologue thereof or to the complement of such anucleic acid strand. A nucleic acid sequence complement of any nucleicacid sequence of the present invention refers to the nucleic acidsequence of the nucleic acid strand that is complementary to (i.e., canform a complete double helix with) the strand for which the sequence iscited. It is to be noted that a double-stranded nucleic acid molecule ofthe present invention for which a nucleic acid sequence has beendetermined for one strand, that is represented by a SEQ ID NO, alsocomprises a complementary strand having a sequence that is a complementof that SEQ ID NO. As such, nucleic acid molecules of the presentinvention, which can be either double-stranded or single-stranded,include those nucleic acid molecules that form stable hybrids understringent hybridization conditions with either a given SEQ ID NO denotedherein and/or with the complement of that SEQ ID NO, which may or maynot be denoted herein. Methods to deduce a complementary sequence areknown to those skilled in the art. Included is a modified annexinnucleic acid molecule that includes a nucleic acid sequence having atleast about 65 percent, preferably at least about 70 percent, morepreferably at least about 75 percent, more preferably at least about 80percent, more preferably at least about 85 percent, more preferably atleast about 90 percent and even more preferably at least about 95percent homology with the corresponding region(s) of the nucleic acidsequence encoding at least a portion of a modified annexin protein.Included is a modified annexin nucleic acid molecule capable of encodinga homodimer of an annexin protein or homologue thereof.

Annexin nucleic acid molecules include SEQ ID NO:4 and allelic variantsof SEQ ID NO:4, SEQ ID NO:1 and an allelic variants of SEQ ID NO:1, SEQID NO:10 and an allelic variants of SEQ ID NO:10; SEQ ID NO:13 and anallelic variants of SEQ ID NO:13; SEQ ID NO:17 and an allelic variantsof SEQ ID NO:17; and SEQ ID NO:21 and an allelic variants of SEQ IDNO:21.

Knowing a nucleic acid molecule of a modified annexin protein of thepresent invention allows one skilled in the art to make copies of thatnucleic acid molecule as well as to obtain a nucleic acid moleculeincluding additional portions of annexin protein-encoding genes (e.g.,nucleic acid molecules that include the translation start site and/ortranscription and/or translation control regions), and/or annexinnucleic acid molecule homologues. Knowing a portion of an amino acidsequence of an annexin protein of the present invention allows oneskilled in the art to clone nucleic acid sequences encoding such anannexin protein. In addition, a desired modified annexin nucleic acidmolecule can be obtained in a variety of ways including screeningappropriate expression libraries with antibodies that bind to annexinproteins of the present invention; traditional cloning techniques usingoligonucleotide probes of the present invention to screen appropriatelibraries or DNA; and PCR amplification of appropriate libraries, or RNAor DNA using oligonucleotide primers of the present invention (genomicand/or cDNA libraries can be used).

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of amodified annexin protein. Oligonucleotides of the present invention canbe RNA, DNA, or derivatives of either. The minimal size of sucholigonucleotides is the size required to form a stable hybrid between agiven oligonucleotide and the complementary sequence on another nucleicacid molecule of the present invention. Minimal size characteristics aredisclosed herein. The size of the oligonucleotide must also besufficient for the use of the oligonucleotide in accordance with thepresent invention. Oligonucleotides of the present invention can be usedin a variety of applications including, but not limited to, as probes toidentify additional nucleic acid molecules, as primers to amplify orextend nucleic acid molecules or in therapeutic applications to modulatemodified annexin production. Such therapeutic applications include theuse of such oligonucleotides in, for example, antisense-, triplexformation-, ribozyme- and/or RNA drug-based technologies. The presentinvention, therefore, includes such oligonucleotides and methods tomodulate the production of modified annexin proteins by use of one ormore of such technologies.

Natural, Wild-type Bacterial Cells and Recombinant Molecules and Cells

The present invention also includes a recombinant vector, which includesa modified annexin nucleic acid molecule of the present inventioninserted into any vector capable of delivering the nucleic acid moleculeinto a host cell. Such a vector contains heterologous nucleic acidsequences, that is, nucleic acid sequences that are not naturally foundadjacent to modified annexin nucleic acid molecules of the presentinvention. The vector can be either RNA or DNA, either prokaryotic oreukaryotic, and typically is a virus or a plasmid. Recombinant vectorscan be used in the cloning, sequencing, and/or otherwise manipulating ofmodified annexin nucleic acid molecules of the present invention. Onetype of recombinant vector, herein referred to as a recombinant moleculeand described in more detail below, can be used in the expression ofnucleic acid molecules of the present invention. Some recombinantvectors are capable of replicating in the transformed cell. Nucleic acidmolecules to include in recombinant vectors of the present invention aredisclosed herein.

As heretofore disclosed, one embodiment of the present invention is amethod to produce a modified annexin protein of the present invention byculturing a cell capable of expressing the protein under conditionseffective to produce the protein, and recovering the protein. In analternative embodiment, the method includes producing an annexin proteinby culturing a cell capable of expressing the protein under conditionseffective to produce the annexin protein, recovering the protein, andmodifying the protein by coupling it to an agent that increases itseffective size.

In one embodiment, the cell to culture is a natural bacterial cell, andmodified annexin is isolated from these cells. In another embodiment, acell to culture is a recombinant cell that is capable of expressing themodified annexin protein, the recombinant cell being produced bytransforming a host cell with one or more nucleic acid molecules of thepresent invention. Transformation of a nucleic acid molecule into a cellcan be accomplished by any method by which a nucleic acid molecule canbe inserted into the cell. Transformation techniques include, but arenot limited to, transfection, electroporation, microinjection,lipofection, adsorption, and protoplast fusion. A recombinant cell mayremain unicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Nucleic acidmolecules with which to transform a host cell are disclosed herein.

Suitable host cells to transform include any cell that can betransformed and that can express the introduced modified annexinprotein. Such cells are, therefore, capable of producing modifiedannexin proteins of the present invention after being transformed withat least one nucleic acid molecule of the present invention. Host cellscan be either untransformed cells or cells that are already transformedwith at least one nucleic acid molecule. Suitable host cells of thepresent invention can include bacterial, fungal (including yeast),insect, animal, and plant cells. Host cells include bacterial cells,with E. coli cells being particularly preferred. Alternative host cellsare untransformed (wild-type) bacterial cells producing cognate modifiedannexin proteins, including attenuated strains with reducedpathogenicity, as appropriate.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase “operatively linked” refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence that is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those that control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to the art. Transcriptioncontrol sequences include those which function in bacterial, yeast,insect and mammalian cells, such as, but not limited to, tac, lac, tzp,trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (λ) (such as λp_(L)and λp_(R) and fusions that include such promoters), bacteriophage T7,T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus,poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral longterminal repeat, Rous sarcoma virus, heat shock, phosphate and nitratetranscription control sequences as well as other sequences capable ofcontrolling gene expression in prokaryotic or eukaryotic cells.Additional suitable transcription control sequences includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).Transcription control sequences of the present invention can alsoinclude naturally occurring transcription control sequences naturallyassociated with a DNA sequence encoding an annexin protein. Onetranscription control sequence is the Kozak strong promotor andinitiation sequence.

Expression vectors of the present invention may also contain secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed annexin protein to be secreted from the cell that produces theprotein. Suitable signal segments include an annexin protein signalsegment or any heterologous signal segment capable of directing thesecretion of an annexin protein, including fusion proteins, of thepresent invention. Signal segments include, but are not limited to,tissue plasminogen activator (t-PA), interferon, interleukin, growthhormone, histocompatibility and viral envelope glycoprotein signalsegments.

Expression vectors of the present invention may also contain fusionsequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of a modified annexin nucleic acid molecule ofthe present invention can enhance the stability during production,storage and/or use of the protein encoded by the nucleic acid molecule.Furthermore, a fusion segment can function as a tool to simplifypurification of a modified annexin protein, such as to enablepurification of the resultant fusion protein using affinitychromatography. One fusion segment that can be used for proteinpurification is the 8-amino acid peptide sequenceasp-tyr-lys-asp-asp-asp-asp-lys (SEQ ID NO:9).

A suitable fusion segment can be a domain of any size that has thedesired function (e.g., increased stability and/or purification tool).It is within the scope of the present invention to use one or morefusion segments. Fusion segments can be joined to amino and/or carboxyltermini of an annexin protein. Another type of fusion protein is afusion protein wherein the fusion segment connects two or more annexinproteins or modified annexin proteins. Linkages between fusion segmentsand annexin proteins can be constructed to be susceptible to cleavage toenable straightforward recovery of the annexin or modified annexinproteins. Fusion proteins are preferably produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of an annexin protein.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecules in the cell to be transformed. A recombinant molecule includesone or more nucleic acid molecules of the present invention, includingthose that encode one or more modified annexin proteins. Recombinantmolecules of the present invention and their production are described inthe Examples section. Similarly, a recombinant cell includes one or morenucleic acid molecules of the present invention, with those that encodeone or more annexin proteins. Recombinant cells of the present inventioninclude those disclosed in the Examples section.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

In accordance with the present invention, recombinant cells can be usedto produce annexin or modified annexin proteins of the present inventionby culturing such cells under conditions effective to produce such aprotein, and recovering the protein. Effective conditions to produce aprotein include, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate, or effective, medium refers to any medium in which a cellof the present invention, when cultured, is capable of producing anannexin or modified annexin protein. Such a medium is typically anaqueous medium comprising assimilable carbohydrate, nitrogen andphosphate sources, as well as appropriate salts, minerals, metals andother nutrients, such as vitamins. The medium may comprise complex,nutrients or may be a defined minimal medium.

Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantannexin proteins may either remain within the recombinant cell; besecreted into the fermentation medium; be secreted into a space betweentwo cellular membranes, such as the periplasmic space in E. coli; or beretained on the outer surface of a cell or viral membrane. Methods topurify such proteins are disclosed in the Examples section.

Antibodies

The present invention also includes isolated anti-modified annexinantibodies and their use. An anti-modified annexin antibody is anantibody capable of selectively binding to a modified annexin protein.Isolated antibodies are antibodies that have been removed from theirnatural milieu. The term “isolated” does not refer to the state ofpurity of such antibodies. As such, isolated antibodies can includeanti-sera containing such antibodies, or antibodies that have beenpurified to varying degrees. As used herein, the term “selectively bindsto” refers to the ability of such antibodies to preferentially bind tothe protein against which the antibody was raised (i.e., to be able todistinguish that protein from unrelated components in a mixture).Binding affinities, commonly expressed as equilibrium associationconstants, typically range from about 10³ M⁻¹ to about 10¹² M⁻¹. Bindingcan be measured using a variety of methods known to those skilled in theart including immunoblot assays, immunoprecipitation assays,radioimmunoassays, enzyme immunoassays (e.g., ELISA), immunofluorescentantibody assays and immunoelectron microscopy; see, e.g., Sambrook etal., 1989.

Antibodies of the present invention can be either polyclonal ormonoclonal antibodies. Antibodies of the present invention includefunctional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein used to obtain the antibodies. Antibodies of the presentinvention also include chimeric antibodies that can bind to more thanone epitope. Antibodies are raised in response to proteins that areencoded, at least in part, by a modified annexin nucleic acid moleculeof the present invention.

Anti-modified annexin antibodies of the present invention includeantibodies raised in an animal administered a modified annexin.Anti-modified annexin antibodies of the present invention also includeantibodies raised in an animal against one or more modified annexinproteins of the present invention that are then recovered from the cellusing techniques known to those skilled in the art. Yet additionalantibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed for modified annexin proteins of thepresent invention. Antibodies produced against defined proteins can beadvantageous because such antibodies are not substantially contaminatedwith antibodies against other substances that might otherwise causeinterference in a diagnostic assay or side effects if used in atherapeutic composition.

Anti-modified annexin antibodies of the present invention have a varietyof uses that are within the scope of the present invention.Anti-modified annexin antibodies can be used as tools to screenexpression libraries and/or to recover desired proteins of the presentinvention from a mixture of proteins and other contaminants.

An anti-modified annexin antibody of the present invention canselectively bind to a modified annexin protein.

Therapeutic Methods

Any of the above-described modified annexin proteins is used in methodsof the invention to treat arterial or venous thrombosis caused by anymedical procedure or condition. Generally, the therapeutic agents usedin the invention are administered to an animal in an effective amount.Generally, an effective amount is an amount effective either (1) toreduce the symptoms of the disease sought to be treated or (2) to inducea pharmacological change relevant to treating the disease sought to betreated.

For thrombosis, an effective amount includes an amount effective toexert prolonged antithrombotic activity without substantially increasingthe risk of hemorrhage or to increase the life expectancy of theaffected animal. As used herein, prolonged antithrombotic activityrefers to the time of activity of the modified annexin protein withrespect to the time of activity of the same amount (molar) of anunmodified annexin protein. Preferably, antithrombotic activity isprolonged by at least about a factor of two, more preferably by at leastabout a factor of five, and most preferably by at least about a factorof ten. Preferably, the effective amount does not substantially increasethe risk of hemorrhage compared with the hemorrhage risk of the samesubject to whom the modified annexin has not been administered.Preferably, the hemorrhage risk is very small and, at most, below thatprovided by alternative antithrombotic treatments available in the priorart. Therapeutically effective amounts of the therapeutic agents can beany amount or dose sufficient to bring about the desired antithromboticeffect and depends, in part, on the condition, type, and location of thethrombus, the size and condition of the patient, as well as otherfactors known to those skilled in the art. The dosages can be given as asingle dose, or as several doses, for example, divided over the courseof several weeks.

Administration preferably occurs by bolus injection or by intravenousinfusion, either after thrombosis to prevent further thrombosis or underconditions in which the subject is susceptible to or at risk ofthrombosis.

The therapeutic agents of the present invention can be administered byany suitable means, including, for example, parenteral or localadministration, such as intravenous or subcutaneous injection, or byaerosol. A therapeutic composition can be administered in a variety ofunit dosage forms depending upon the method of administration. Deliverymethods for a therapeutic composition of the present invention includeintravenous administration and local administration by, for example,injection. For particular modes of delivery, a therapeutic compositionof the present invention can be formulated in an excipient of thepresent invention. A therapeutic agent of the present invention can beadministered to any animal, preferably to mammals, and more preferablyto humans.

One suitable administration time occurs following coronary thrombosis,thereby preventing the recurrence of thrombosis without substantiallyincreasing the risk of hemorrhage. Bolus injection of the modifiedannexin is preferably performed soon after thrombosis, e.g., beforeadmission to hospital. The modified annexin can be administered inconjunction with a thrombolytic therapeutic such as tissue plasminogenactivator, urokinase, or a bacterial enzyme.

Methods of use of modified annexin proteins of the present inventioninclude methods to treat cerebral thrombosis, including overt cerebralthrombosis or transient cerebral ischemic attacks, by administering aneffective amount of modified annexin protein to a patient in needthereof. Transient cerebral ischemic attacks frequently precedefull-blown strokes. The modified annexin can also be administered todiabetic and other patients who are at increased risk for thrombosis inperipheral arteries. Accordingly, the present invention provides amethod for reducing the risk of thrombosis in a patient having anincreased risk for thrombosis including administering an effectiveamount of a modified annexin protein to a patient in need thereof. Foran adult patient, the modified annexin can be administered intravenouslyor as a bolus in the dosage range of about 1 to about 100 mg.

The present invention also provides a method for decreasing the risk ofvenous thrombosis associated with some surgical procedures, such as hipand knee arthroplasties, by administering an effective amount of amodified annexin protein of the present invention to a patient in needthereof. The modified annexin treatment can prevent thrombosis withoutincreasing hemorrhage into the operating field. In another embodiment,the present invention provides a method for preventing thrombosisassociated with pregnancy and parturition without increasing hemorrhage,by administering an effective amount of a modified annexin protein ofthe present invention to a patient in need thereof. In a furtherembodiment, the present invention provides a method for the treatment ofrecurrent venous thrombosis, by administering an effective amount of amodified annexin protein of the present invention to a patient in needthereof. For an adult patient, the modified annexin can be administeredintravenously as a bolus in the dosage range of about 1 to about 100 mg.

The present invention also provides a method of screening for a modifiedannexin protein that modulates thrombosis, by contacting a thrombosistest system with at least one test modified annexin protein underconditions permissive for thrombosis, and comparing the antithromboticactivity in the presence of the test modified annexin protein with theantithrombotic activity in the absence of the test modified annexinprotein, wherein a change in the antithrombotic activity in the presenceof the test modified annexin protein is indicative of a modified annexinprotein that modulates thrombotic activity. In one embodiment, thethrombosis test system is a system for measuring activated partialthromboplastin time. Also included within the scope of the presentinvention are modified annexin proteins that modulate thrombosis asidentified by this method.

The present invention also provides a method for identifying a modifiedannexin protein for annexin activity, including contacting activatedplatelets with at least one test modified annexin protein underconditions permissive for binding, and comparing the test modifiedannexin-binding activity and protein S-binding activity of the plateletsin the presence of the test modified annexin protein with theannexin-binding activity and protein S-binding activity in the presenceof unmodified annexin protein, whereby a modified annexin protein withannexin activity may be identified. Also included within the scope ofthe invention are modified annexin proteins identified by the method.

In an additional embodiment, the present invention provides a method ofscreening for a modified annexin protein that modulates thrombosis, bycontacting an in vivo thrombosis test system with at least one testmodified annexin protein under conditions permissive for thrombosis, andcomparing the antithrombotic activity in the presence of the testmodified annexin protein with the antithrombotic activity in the absenceof the test modified annexin protein. A change in the antithromboticactivity in the presence of the test modified annexin protein isindicative of a modified annexin protein that modulates thromboticactivity. Additionally, the time over which antithrombotic activity issustained in the presence of the test modified annexin protein iscompared with a time of antithrombotic activity in the presence ofunmodified annexin to determine the prolongation of antithromboticactivity associated with the test modified annexin protein. The extentof hemorrhage in the presence of the test modified annexin protein isassessed, e.g., by measuring tail bleeding time, and compared with theextent of hemorrhage in the absence of the test modified annexinprotein. In one embodiment, the in vivo thrombosis test system is amouse model of photochemically-induced thrombus in cremaster muscles.Also included within the scope of the present invention are modifiedannexin proteins that modulate thrombosis as identified by this method.

In a further embodiment, the therapeutic agents of the present inventionare useful for gene therapy. As used herein, the phrase “gene therapy”refers to the transfer of genetic material (e.g., DNA or RNA) ofinterest into a host to treat or prevent a genetic or acquired diseaseor condition. The genetic material of interest encodes a product (e.g.,a protein polypeptide, peptide or functional RNA) whose production invivo is desired. For example, the genetic material of interest canencode a hormone, receptor, enzyme or (poly)peptide of therapeuticvalue. In a specific embodiment, the subject invention utilizes a classof lipid molecules for use in non-viral gene therapy which can complexwith nucleic acids as described in Hughes et al., U.S. Pat. No.6,169,078, incorporated herein by reference, in which a disulfide linkeris provided between a polar head group and a lipophilic tail group of alipid.

These therapeutic compounds of the present invention effectively complexwith DNA and facilitate the transfer of DNA through a cell membrane intothe intracellular space of a cell to be transformed with heterologousDNA. Furthermore, these lipid molecules facilitate the release ofheterologous DNA in the cell cytoplasm thereby increasing genetransfection during gene therapy in a human or animal.

Cationic lipid-polyanionic macromolecule aggregates may be formed by avariety of methods known in the art. Representative methods aredisclosed by Felgner et al., Proc. Natl. Acad. Sci. USA 86: 7413-7417(1987); Eppstein et al., U.S. Pat. No. 4,897,355; Behr et al., Proc.Natl. Acad. Sci. USA 86:6982-6986 (1989); Bangham et al., J. Mol. Biol.23:238-252 (1965); Olson et al., Biochim. Biophys. Acta 557:9 (1979);Szoka, et al., Proc. Natl. Acad. Sci. 75:4194 (1978); Mayhew et al.,Biochim. Biophys. Acta 775:169 (1984); Kim et al., Biochim. Biophys.Acta 728:339 (1983); and Fukunaga et al., Endocrinol. 115:757 (1984),all incorporated herein by reference. In general, aggregates may beformed by preparing lipid particles consisting of either (1) a cationiclipid or (2) a cationic lipid mixed with a colipid, followed by adding apolyanionic macromolecule to the lipid particles at about roomtemperature (about 18 to 26° C.). In general, conditions are chosen thatare not conducive to deprotection of protected groups. In oneembodiment, the mixture is then allowed to form an aggregate over aperiod of about 10 minutes to about 20 hours, with about 15 to 60minutes most conveniently used. Other time periods may be appropriatefor specific lipid types. The complexes may be formed over a longerperiod, but additional enhancement of transfection efficiency will notusually be gained by a longer period of complexing.

The compounds and methods of the subject invention can be used tointracellularly deliver a desired molecule, such as, for example, apolynucleotide, to a target cell. The desired polynucleotide can becomposed of DNA or RNA or analogs thereof. The desired polynucleotidesdelivered using the present invention can be composed of nucleotidesequences that provide different functions or activities, such asnucleotides that have a regulatory function, e.g., promoter sequences,or that encode a polypeptide. The desired polynucleotide can alsoprovide nucleotide sequences that are antisense to other nucleotidesequences in the cell. For example, the desired polynucleotide whentranscribed in the cell can provide a polynucleotide that has a sequencethat is antisense to other nucleotide sequences in the cell. Theantisense sequences can hybridize to the sense strand sequences in thecell. Polynucleotides that provide antisense sequences can be readilyprepared by the ordinarily skilled artisan. The desired polynucleotidedelivered into the cell can also comprise a nucleotide sequence that iscapable of forming a triplex complex with double-stranded DNA in thecell.

The present invention provides compounds and methods for preventing orattenuating reperfusion injury in mammals. Reperfusion injury (RI)occurs when the blood supply to an organ or tissue is cut off and afteran interval restored. The loss of phospholipid asymmetry in endothelialcells and other cells is considered a significant event in thepathogenesis of RI. The PS exposed on the surfaces of these cells allowsthe binding of activated monocytes. This binding triggers a sequence ofevents leading to irreversible apoptosis of endothelial and other cells,another significant event in RI. In addition, PS on the surfaces ofcells, and vesicles derived therefrom, is accessible to phospholipasesthat generate lipid mediators. These lipid mediators amplify the damageoccuffing by mechanisms described above and produce seriouscomplications such as ventricular arrhythmia following acute myocardialinfarction.

A recombinant human annexin, preferably annexin V, is modified in such away that its half-life in the vascular compartment is prolonged. Thiscan be achieved in a variety of ways; three embodiments are an annexincoupled to polyethylene glycol, a homopolymer or heteropolymer ofannexin, and a fusion protein of annexin with another protein (e.g., theFc portion of immunoglobulin). See Allison, “Modified Annexin Proteinsand Methods for Preventing Thrombosis,” U.S. patent application Ser. No.10/080,370 (filed Feb. 21, 2002 , now U.S. Patent No. 6,962,903) andAllison, “Modified Annexin Proteins and Methods for TreatingVaso-Occlusive Sickle-Cell Disease,” U.S. patent application Ser. No.10/632,694 (filed Aug. 1, 2003, now U.S. Pat. No. 6,982,154), bothincorporated by reference herein in their entirety.

The modified annexin binds with high affinity to phosphatidylserine onthe surface of epithelial and other cells, thereby preventing thebinding of phagocytes and the operation of phospholipases, which releaselipid mediators. The modified annexin therefore inhibits both cellularand humoral mechanisms of reperfusion injury.

In one embodiment, the present invention provides an isolated modifiedannexin protein containing an annexin protein coupled to at least oneadditional protein, such as an additional annexin protein (forming ahomodimer), polyethylene glycol, or the Fc portion of immunoglobulin.The additional protein preferably has a molecular weight of at least 30kDa. Also provided by the present invention are pharmaceuticalcompositions containing an amount of any of the modified annexinproteins of the invention that is effective for preventing or reducingreperfusion injury.

In some methods of the invention, the modified annexin is administeredto a subject at risk of reperfusion injury in a pharmaceuticalcomposition having an amount of any one of the modified annexin proteinsof the present invention effective for preventing or attenuatingreperfusion injury. For example, the pharmaceutical composition may beadministered before and after organ transplantation, arthroplasty orother surgical procedure in which the blood supply to organ or tissue iscut off and after an interval restored. It can also be administeredafter a coronary or cerebral thrombosis.

The modified annexin binds PS accessible on cell surfaces (shielding thecells), thereby preventing the attachment of monocytes and theirreversible stage of apoptosis. In addition, the modified annexininhibits the activity of phospholipases that generate lipid mediatorsthat also contribute to RI. The modified annexin will be useful toprevent or attenuate RI and protect organs in organs transplanted fromcadaver donors, in patients with coronary and cerebral thrombosis, inpatients undergoing arthroplasties, and in other situations. In additionthe modified annexin will exert prolonged antithrombotic activitywithout increasing hemorrhage. This combination of antithromboticpotency with capacity to attenuate RI presents a unique profile ofdesirable activities not displayed by any therapeutic agent currentlyused or known to be in development.

As described in Example 6, the annexin homodimer is a potent inhibitorof sPLA₂ (FIG. 4). Because annexin V binds to PS on cell surfaces withhigh affinity, it shields PS from degradation by sPLA₂ and otherphospholipases.

Producing a homodimer of human annexin V both increased its affinity forPS, thereby improving its efficacy as a therapeutic agent; and augmentedits size, thereby prolonging its survival in the circulation andduration of action. The 36 kDa monomer is lost rapidly from the bloodstream into the kidneys. In the rabbit more than 80% of labeled annexinV injected into the circulation disappears in 7 minutes (Thiagarajan andBenedict, Circulation 96: 2339, 1997). In cynomolgus monkeys thehalf-life of injected annexin V was found to be 11 to 15 minutes(Romisch et al., Thrombosis Res., 61: 93, 1991). In humans injected withannexin V labeled with 99MTc, the half-life with respect to the major(α) compartment was 24 minutes (Kemerink et al., J. Nucl. Med. 44: 947,2003).

The annexin homodimer may be produced by any convenient method. In someembodiments, the annexin homodimer is produced by recombinant DNAtechnology as this avoids the necessity for post-translation proceduressuch as linkage to the one available sulfhydryl group in the monomer orcoupling with polyethylene glycol. Recombinant homodimerization wasachieved by the use of a flexible peptide linker attached to the aminoterminus of one annexin monomer and the carboxy terminus of the other(FIG. 1). The three-dimensional structure of annexin V, and the residuesbinding Ca²⁺and PS, are known from X-ray crystallography andsite-specific mutagenesis (Huber et al., J. Mol. Biol. 223: 683, 1992;Campos et al., 37: 8004, 1998). The Ca²⁺- and PS-binding sites are onthe convex surface of the molecule while the amino terminus forms aloose tail on the concave surface. The annexin V homodimer shown in FIG.1 is designed so that the convex surfaces could fold in such a way thatboth could gain access to PS on cell surfaces. Thus, for this reason,the dimer would have a higher affinity for PS than that of the monomer.As reported in Example 4, this was verified experimentally. Anotheradvantage of the homodimer of annexin V is that while a molecule of 36kDa (the monomer) would be lost rapidly from circulation into thekidney, one of 73 kDa (the dimer), exceeding the renal filtrationthreshold, would not. Hence, the therapeutically useful activity wouldbe prolonged in the dimer. This prediction was confirmed in experiments.

To prevent or attenuate reinfarction and RI, it is desirable, in someinstances, to have a longer duration of activity. Increasing themolecular weight of annexin V by homodimerization to 76 kDa preventsrenal loss and extends survival in the circulation. Accordingly, suchmodified annexins may effectively attenuate RI, even when administeredseveral hours before the blood supply to an organ is cut off.

The teachings of the present invention are contrary to reports in theliterature suggesting that annexin V does not inhibit RI. For example,d'Amico et al. report that annexin V did not inhibit RI in the rat heartwhereas lipocortin I (annexin I) did (d'Amico et al., FASEB J. 14: 1867,2000). A fragment of lipocortin I, injected into the cerebral ventricleof rats, was reported to decrease infarct size and cerebral edema aftercerebral ischemia (Pelton et al., J. Exp. Med. 174: 305, 1991); theseauthors did not study reperfusion. In a comprehensive review ofstrategies to prevent ischemic injury of the liver (Selzner et al.,Gastroenterology 15:917, 2003), annexin is not mentioned.

As described in Example 7, the ability of the annexin V homodimer toattenuate RI was tested in a mouse liver model (Teoh et al., Hepatology36:94, 2002). In this model, the blood supply to the left lateral andmedian lobes of the liver is cut off for 90 minutes and then restored.After 24 hours, the severity of liver injury is assessed by serum levelsof alanine aminotransferase (ALT) and hepatic histology. Both theannexin V homodimer (DAV), molecular weight 73kDa, and annexin V coupledto polyethylene glycol (PEG-AV), molecular weight 57 kDa, injected 6hours before clamping the hepatic arteries, were highly effective inattenuating RI as shown by serum ALT levels (FIG. 5) and hepatichistology. The annexin V monomer (AV) was less protective in this model.In Example 13, a similar procedure was performed in which the annexin Vhomodimer was administered at 10 minutes and 60 minutes after thecommencement of reperfusion. Similar protection against IRI is found.

The experimental evidence therefore confirms that the modified annexinsof the present invention will be useful to attenuate RI in subjects. Asdiscussed above, similar pathogenetic mechanisms are involved in theforms of RI occurring in different organs, thus, the annexin V homodimermay be used to attenuate RI in all of them.

Because of its high affinity for PS and reduced loss from thecirculation, the annexin V homodimer will exert prolonged antithromboticactivity. This is clinically useful to prevent reinfarction, which isknown to be an important event following coronary thrombosis (Andersenet al., N. Engl. J. Med. 349: 733, 2003), and is likely to be importantin stroke. Prevention of thrombosis in patients undergoing arthroplastyis also a major clinical need. The additional activity of a modifiedannexin as an anticoagulant is therefore valuable. In severalexperimental animal models, annexin V inhibits arterial and venousthrombosis without increasing hemorrhage (Römisch et al., Thromb. Res.61: 93, 1991; Van Ryn-McKenna et al., Thromb. Hemost. 69: 227, 1993;Thiagarajan and Benedict, Circulation 96: 2339, 1997). A modifiedannexin has the capacity to exert anticoagulant activity withoutincreasing hemorrhage and to attenuate reperfusion injury. Thiscombination of actions could be useful in several clinical situations.No other therapeutic agent currently used, or known to be indevelopment, shares this desirable profile of activities.

Several annexins, other than annexin V, bind Ca² + and PS. Any of thesemight be used to prevent or diminish reperfusion injury. The molecularweight of annexin V, or another annexin, may be increased by proceduresother than homodimerization. Such procedures include the preparation ofother homopolymers or heteropolymers. Alternatively, an annexin might beconjugated to another protein by recombinant DNA technology or chemicalmanipulation. Conjugation of an annexin to polyethylene glycol oranother nonpeptide compound are also envisaged.

It is expected that the annexin V homodimer will be well-tolerated.Another annexin, annexin VI, is a naturally existing homodimer of theconserved annexin sequence. However, annexin VI does not bind PS withhigh affinity A PS-binding protein other than an annexin may also beused in the methods of the invention. For example, a monoclonal orpolyclonal antibody with a high affinity for PS (Diaz et al.,Bioconjugate Chem. 9:250, 1998; Thorpe et al., U.S. Pat. No. 6,312,694)may be used according to the present invention (e.g., for decreasing orpreventing reperfusion injury).

Diannexin (SEQ ID NO: 27) has dose-related antithrombotic activity inthe rat (FIG. 7). In contrast, Fragmin (low molecular weight heparin)administered at 140 aXa units/kg (approx. 7x therapeutic dose)significantly increased blood loss in experiments conductedsimultaneously (Table 4 and FIG. 10). Regarding the APTT (activatedprothrombin time), none of the doses of Diannexin used increased theAPTT, whereas both 20 aXa units/kg (Table 2) of Fragmin, and 140 aXaunits/kg (Table 5 and FIG. 11) significantly increased the APTT.Clearance of iodine-labeled Diannexin could be described by atwo-compartment model, an α-phase of 9-14 min and a β-phase of 6-7 hrs(FIG. 12). The latter is significantly longer than previously reportedfor annexin IV monomer in several species. The 6.5 hour half life isconvenient therapeutically because a single bolus injection shouldsuffice for many clinical applications of Diannexin. In the unlikelyevent that Diannexin induces hemorrhage its effects will disappearfairly soon. Both Diannexin and Fragmin significantly increase thebleeding time in the rat following tail transection (FIG. 9 and Table4). In the case of Diannexin this may be due to inhibition ofphospholipase A₂ action and thromboxane generation. In humans bleedingtimes are increased when cyclooxygenase is inhibited by a drug or as aresult of a genetic deficiency. Diannexin administration has no effecton body weight.

The present invention provides compounds and methods for preventing orattenuating cold ischemia-warm reperfusion injury in mammals. Asdescribed above, organs to be used for transplantation are typicallyrecovered from cadaver donors and perfused with a saline solution suchas the University of Wisconsin solution originally introduced by Belzeret al. (Transplantation 1988; 45: 673). The organs are then preserved onice for several hours before being transplanted. During this period theorgan is anoxic, which results in depletion of ATP and loss ofphospholipid asymmetry in the plasma membranes of endothelial cells (EC)and other cells. Under normal conditions an ATP-dependent phospholipidtranslocase maintains this asymmetry, which confines PS to the innerleaflet of the plasma membrane bilayer. Following anoxia PS isdemonstrable on the outer leaflet of the EC plasma membrane, as shown byannexin V binding to the surface of cultured cells (Ran et al. CancerRes. 2002; 62: 6132) Generally, the present invention comprises a methodof protecting organs or tissue susceptible to IRI, wherein said organsor tissue are contacted with a modified annexin protein. Thus, theorgans or tissue can be contacted with a modified annexin protein byparenterally administering about 10 to 1000 μg/kg of modified annexinprotein to a patient who has organs or tissue susceptible to a conditionof IRI, even in the case of donors with fatty livers. In someembodiments, the modified annexin protein is administered in a range ofabout 100 to 500 μg/kg. Modified annexin proteins are shown herein toattenuate IRI in organ transplantation, even in the case of patient witha fatty liver. The ability to attenuate IRI in the case of a steatoticliver transplant will increase the number of livers considered suitablefor use. The present invention therefore has utility as the number ofpatients who would benefit from liver transplantation greatly exceedsthe number of organs available.

In another embodiment of the invention, to protect organ transplants,modified annexin proteins can be added to the preservation fluid usedfor in situ organ perfusion and cooling in the donor and for coldstorage or perfusion after the organ is harvested. The organ or tissuetransplants can be perfused or flushed with a solution containingmodified annexin proteins in a concentration of 0.1 to 1 mg/l.Typically, the organs or tissue are perfused with a solution containing,in addition to modified annexin proteins, components such aselectrolytes and cell-protecting agents. According to the presentinvention, a modified annexin, such as SEQ ID NO:6, SEQ ID NO:19, or SEQID NO:23 is used.

In summary, when used for treating patients, modified annexin proteinsare, according to the present invention, administered intravenously,subcutaneously, or by other suitable route. In addition we havedemonstrated that when Diannexin is added to the University of Wisconsinsolution perfusing rat livers ex vivo after recovery, before overnightstorage at 4° and just before transplantation, it is also effective inpreventing IRI and protecting organs in recipients. This provides analternative or supplementary method of administration when Diannexin isused to prevent IRI and protect the organ in liver graft recipients.Addition of Diannexin to the fluid perfusing kidneys, hearts and otherorgans may also decrease IR following transplantation.

Turning now to the use of modified annexin proteins in preservation orrinse solutions it can be reiterated that by adding modified annexinproteins to the preservation solution used for organ perfusion andcooling in the donor and for cold storage or perfusion after the organis harvested, IR injury in the organ transplant can be prevented andfunctional recovery after transplantation promoted. Modified annexinproteins may be added to different types of preservation solutions,which typically contain electrolytes (such as Na⁺, K⁺, Mg⁺⁺, Cl^(−;),SO₄ ^(2−;), HPO₄ ^(2−;), Ca²⁺ and HCO₃ ^(−;)) and may contain variousother agents protecting the cells during cold storage. For example, AGPand/or AAT can be added to the University of Wisconsin Belzer solutionwhich contains 50 g/l hydroxyethyl starch, 35.83 g/l lactobionic acid,3.4 g/l potassium phosphate monobasic, 1.23 g/l magnesium sulfateheptahydrate, 17.83 g/l raffinose pentahydrate, 1.34 g/l adenosine,0.136 g/l allopurinol, 0.922 g/l glutathionine, 5.61 g/l potassiumhydroxide and sodium hydroxide for adjustment of pH to pH 7.4. Anotherexample of a suitable preservation solution is the Euro-Collinssolution, which contains 2.05 g/l mono-potassium phosphate, 7.4 g/ldipotassium phosphate, 1.12 g/l potassium chloride, 0.84 g/l sodiumbicarbonate and 35 g/l glucose. These intracellular type preservationsolutions are rinsed away from the donor organ before completion oftransplantation into the recipient by using a physiological infusionsolution, such as Ringer's solution, and modified annexin proteins canbe also added to a rinse solution. Further, modified annexin proteinscan be added to extracellular type preservation solutions which need tobe flushed away, such as PEFADEX (Vitrolife, Sweden), which contains 50g/l dextran, 8 g/l sodium chloride, 400 mg/l potassium chloride, 98 mg/lmagnesium sulfate, 46 mg/l disodium phosphate, 63 mg/l potassiumphosphate and 910 mg/l glucose.

The novel preservation and rinsing solutions according to the presentinvention may have a composition essentially corresponding to any of thethree commercial solutions described above. However, the actualconcentrations of the conventional components may vary somewhat,typically within a range of about ±50%, preferably about ±30%, of themean values given above.

In one embodiment, to ensure maximum activity, modified annexin proteinsare added to a ready-made preservation or rinse solution just beforeuse. Alternatively, a suitable preservation solution containing modifiedannexin proteins may be prepared beforehand.

By administrating modified annexin proteins to patients undergoingcardiac or angioplastic surgery, development of IR injury following theoperation can be prevented and the heart can be protected. Thisdecreases the need of postoperative critical care. Correspondingly, byadministering modified annexin proteins to patients undergoingthrombolytic therapy, development of IR injury during reperfusion of theoccluded vessel can be prevented and organ dysfunction can be avoided.In thrombolytic therapy of myocardial infarction this may preventcardiac arrythmias and cardiac insufficiency. In thrombolytic therapy ofbrain infarction, this may decrease neurological symptoms and palsies.By administrating modified annexin proteins to patients suffering frombleeding shock, septic shock, or other forms of shock, development of IRinjury can be prevented.

By administrating modified annexin proteins to patients undergoingcardiac or angioplastic surgery, development of IR injury following theoperation can be prevented and the heart can be protected. Thisdecreases the need of postoperative critical care. Correspondingly, byadministering modified annexin proteins to patients undergoingthrombolytic therapy, development of IR injury during reperfusion of theoccluded vessel can be prevented and organ dysfunction can be avoided.In thrombolytic therapy of myocardial infarction this may preventcardiac arrhythmias and cardiac insufficiency. In thrombolytic therapyof brain infarction, this may decrease neurological symptoms andpalsies. By administrating modified annexin proteins to patientssuffering from bleeding shock, septic shock, or other forms of shock,development of IR injury can be prevented.

According to an embodiment of the present invention, modified annexinproteins and mixtures thereof are used in methods for preparingpharmaceutical compositions intended for use in any of the therapeuticmethods of treatment described above.

The present invention is also directed toward therapeutic compositionscomprising the modified annexin proteins of the present invention.Compositions of the present invention can also include other componentssuch as a pharmaceutically acceptable excipient, an adjuvant, and/or acarrier. For example, compositions of the present invention can beformulated in an excipient that the animal to be treated can tolerate.Examples of such excipients include water, saline, Ringer's solution,dextrose solution, mannitol, Hanks' solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such astriglycerides may also be used. Excipients can also contain minoramounts of additives, such as substances that enhance isotonicity andchemical stability. Examples of buffers include phosphate buffer,bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, ormixtures thereof, while examples of preservatives include thimerosal, m-or o-cresol, formalin and benzyl alcohol. Standard formulations caneither be liquid injectables or solids which can be taken up in asuitable liquid as a suspension or solution for injection. Thus, in anon-liquid formulation, the excipient can comprise dextrose, human serumalbumin, preservatives, etc., to which sterile water or saline can beadded prior to administration.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

Generally, the therapeutic agents used in the invention are administeredto an animal in an effective amount. Generally, an effective amount isan amount effective to either (1) reduce the symptoms of the diseasesought to be treated or (2) induce a pharmacological change relevant totreating the disease sought to be treated.

Therapeutically effective amounts of the therapeutic agents can be anyamount or doses sufficient to bring about the desired effect and depend,in part, on the condition, type and location of the thrombosis, the sizeand condition of the patient, as well as other factors readily known tothose skilled in the art. The dosages can be given as a single dose, oras several doses, for example, divided over the course of several days.

The present invention is also directed toward methods of treatmentutilizing the therapeutic compositions of the present invention. Themethod comprises administering the therapeutic agent to a subject inneed of such administration.

The therapeutic agents of the instant invention can be administered byany suitable means, including, for example, parenteral, topical, oral orlocal administration, such as intradermally, by injection, or byaerosol. In one embodiment of the invention, the agent is administeredby injection. Such injection can be locally administered to any affectedarea. A therapeutic composition can be administered in a variety of unitdosage forms depending upon the method of administration. Suitabledelivery methods for a therapeutic composition of the present inventioninclude intravenous administration and local administration by, forexample, injection or introduction into an intravenous drip. Forparticular modes of delivery, a therapeutic composition of the presentinvention can be formulated in an excipient. A therapeutic reagent ofthe present invention can be administered to any animal, preferably tomammals, and more preferably to humans.

The particular mode of administration will depend on the condition to betreated. It is contemplated that administration of the agents of thepresent invention may be via any bodily fluid, or any target or anytissue accessible through a body fluid.

Examples of such fluid include blood and blood products. In livertransplantation, which is included in the present invention,thrombocytopenia is common and blood platelets are transfused. Thesurvival of blood platelets may be improved by co-administration of anannexin or modified annexin such as Diannexin. Stored platelets oftenexpress phosphatidylserine on their surfaces, facilitating attachment toone another on to monocyte-macrophage lineage cells. An annexin couldmask PS on the surface of platelets, thereby improving their survivalduring storage and in patients. Accordingly, the present inventionprovides a method of increasing the duration of survival of bloodplatelets, comprising adding an isolated annexin protein to storedplatelets. The isolated annexin protein may be modified, and in someembodiments may be an annexin dimer. The addition may be in a plateletstorage medium. The addition may also be in a patient to whom plateletsare administered, including the case where the patient is the recipientof a liver graft, including a thrombocytopenic liver graft patient.

The following examples illustrate the preparation of modified annexinproteins of the invention and in vitro and in vivo assays foranticoagulant activity of modified annexin proteins. It is to beunderstood that the invention is not limited to the exemplary workdescribed or to the specific details set forth in the examples.

EXAMPLES Example 1 Modified Annexin Preparation

A. PEGylated Annexins. Annexins can be purified from human tissues orproduced by recombinant technology. For instance, annexin V can bepurified from human placentas as described by Funakoshi et al. (1987).Examples of recombinant products are the expression of annexin II andannexin V in Escherichia coli (Kang, H.-M., Trends Cardiovasc. Med.9:92-102 (1999); Thiagarajan and Benedict, 1997, 2000). A rapid andefficient purification method for recombinant annexin V, based onCa²⁺-enhanced binding to phosphatidylserine-containing liposomes andsubsequent elution by EDTA, has been described by Berger, FEBS Lett.329:25-28 (1993). This procedure can be improved by the use ofphosphatidylserine coupled to a solid phase support.

Annexins can be coupled to polyethylene glycol (PEG) by any of severalwell-established procedures (reviewed by Hermanson, 1996) in a processreferred to as pegylation. The present invention includeschemically-derivatized annexin molecules having mono- or poly-(e.g.,2-4) PEG moieties. Methods for preparing a pegylated annexin generallyinclude the steps of (a) reacting the annexin with polyethylene glycol(such as a reactive ester or aldehyde derivative of PEG) underconditions whereby the annexin becomes attached to one or more PEGgroups and (b) obtaining the reaction product or products. In general,the optimal reaction conditions for the reactions must be determinedcase by case based on known parameters and the desired result.Furthermore, the reaction may produce different products having adifferent number of PEG chains, and further purification may be neededto obtain the desired product.

Conjugation of PEG to annexin V can be performed using the EDC plussulfo-NHS procedure. EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride) is used to form active ester groups with carboxylategroups using sulfo-NHS (N-hydroxysulfosuccinamide). This increases thestability of the active intermediate, which reacts with an amine to givea stable amide linkage. The conjugation can be carried out as describedin Hermanson, 1996.

Bioconjugate methods can be used to produce homopolymers orheteropolymers of annexin; methods are reviewed by Hermanson, 1996.Recombinant methods can also be used to produce fusion proteins, e.g.,annexin expressed with the Fc. portion of immunoglobulin or anotherprotein. The heterotetramer of annexin II with P11 has also beenproduced in E. coli (Kang et al., 1999). All of these proceduresincrease the molecular weight of annexin and have the potential toincrease the half-life of annexin in the circulation and prolong itsanticoagulant effect.

B. homodimer of annexin V. A homodimer of annexin V can be producedusing a DNA construct shown schematically in FIG. 1C (5′-3′sense strand)(SEQ ID NO:4) and coding for an amino acid sequence represented by SEQID NO:6. In this example, the annexin V gene is cloned into theexpression vector pCMV FLAG 2 (available from Sigma-Aldrich) at EcoRIand Bg1II sites. The exact sequences prior to and after the annexin Vsequence are unknown and denoted as “x”. It is therefore necessary tosequence the construct prior to modification to assure proper codonalignment. The pCMV FLAG 2 vector comes with a strong promotor andinitiation sequence (Kozak) and start site (ATG) built in. The startcodon before each annexin V gene must therefore be removed, and a strongstop for tight expression should be added at the terminus of the secondannexin V gene. The vector also comes with an 8-amino acid peptidesequence that can be used for protein purification(asp-tyr-lys-asp-asp-asp-asp-lys) (SEQ ID NO:9). A 14-amino acid spacerwith glycine-serine swivel ends allows optimal rotation between tandemgene-encoded proteins. Addition of restriction sites PvuII and Scalallow removal of the linker if necessary. Addition of a protease siteallows cleavage of tandem proteins following expression. PreScission™protease is available from Amersham Pharmacia Biotech and can be used tocleave tandem proteins. Two annexin V homodimers were generated. In thefirst, a “His tag” was placed at the amino terminal end of the dimer tofacilitate purification (FIG. 1A). The linker sequence of 12 amino acidswas flanked by a glycine and a serine residue at either end to serve asswivels. The structural scheme is shown in FIG. 1A. The amino acidsequence of the His-tagged annexin V homodimer is provided below:

SEQ ID NO: 26 MHHHHHHQAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDDGSLEVLFQGPSGKLAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKK ALLLLCGEDD

The “swivel” amino acids of the linker are bolded and underlined. ThisHis-tagged annexin V homodimer was expressed at a high level inEscherichia coli and purified using a nickel column. The DNA in theconstruct was shown to have the correct sequence and the dimer had thepredicted molecular weight (74kDa). MALDI-TOF mass spectrometry wasaccomplished using a PerSeptive Biosystems Voyager-DE Pro workstationoperating in linear, positive ion mode with a static acceleratingvoltage of 25 kV and a delay time of 40 nsec.

A second human annexin V homodimer was synthesized without the His tag.The structural scheme is shown in FIG. 1B. The amino acid sequence ofthe (non-His-tagged) annexin V homodimer is provided below:

(SEQ ID NO: 27) MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMI KGDTSGDYKKALLLLCGEDDGS LEVLFQGP SG KLAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCG EDD

Again, the “swivel” amino acids of the linker are bolded and underlined.This dimer was expressed at a high level in E.coli and purified byion-exchange chromatography followed by heparin affinity chromatography.The ion-exchange column was from Bio-Rad (Econo-pak HighQ Support) andthe heparin affinity column was from Amersham Biosciences (HiTrapHeparin HP). Both were used according to manufacturers' instructions.Again, the DNA sequence of the annexin V homodimer was found to becorrect. Mass spectrometry showed a protein of 73kDa, as expected. Theamino acid sequence of annexin and other proteins is routinelydetermined in this laboratory by mass spectrometry of peptide fragments.Expected sequences were obtained.

Human Annexin V has the following amino acid sequence:

(SEQ ID NO:3) AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIK GDTSGDYKKALLLLCGEDD

The nucleotide sequence of human annexin V, inserted as indicated in theDNA construct illustrated in FIG. 1C, is as follows:

(SEQ ID NO:1) GCACAGGTTCTCAGAGGCACTGTGACTGACTTCCCTGGATTTGATGAGCGGGCTGATGCAGAAACTCTTCGGAAGGCTATGAAAGGCTTGGGCACAGATGAGGAGAGCATCCTGACTCTGTTGACATCCCGAAGTAATGCTCAGCGCCAGGAAATCTCTGCAGCTTTTAAGACTCTGTTTGGCAGGGATCTTCTGGATGACCTGAAATCAGAACTAACTGGAAAATTTGAAAAATTAATTGTGGCTCTGATGAAACCCTCTCGGCTTTATGATGCTTATGAACTGAAACATGCCTTGAAGGGAGCTGGAACAAATGAAAAAGTACTGACAGAAATTATTGCTTCAAGGACACCTGAAGAACTGAGAGCCATCAAACAAGTTTATGAAGAAGAATATGGCTCAAGCCTGGAAGATGACGTGGTGGGGGACACTTCAGGGTACTACCAGCGGATGTTGGTGGTTCTCCTTCAGGCTAACAGAGACCCTGATGCTGGAATTGATGAAGCTCAAGTTGAACAAGATGCTCAGGCTTTATTTCAGGCTGGAGAACTTAAATGGGGGACAGATGAAGAAAAGTTTATCACCATCTTTGGAACACGAAGTGTGTCTCATTTGAGAAAGGTGTTTGACAAGTACATGACTATATCAGGATTTCAAATTGAGGAAACCATTGACCGCGAGACTTCTGGCAATTTAGAGCAACTACTCCTTGCTGTTGTGAAATCTATTCGAAGTATACCTGCCTACCTTGCAGAGACCCTCTATTATGCTATGAAGGGAGCTGGGACAGATGATCATACCCTCATCAGAGTCATGGTTTCCAGGAGTGAGATTGATCTGTTTAACATCAGGAAGGAGTTTAGGAAGAATTTTGCCACCTCTCTTTATTCCATGATTAAGGGAGATACATCTGGGGACTATAAGAAAGCTCTTCTGCTGCTCTGTGGAGA AGATGAC

C. Annexin IV Homodimer. A homodimer of annexin IV was preparedsimilarly to the annexin V homodimer described in Example 1B. The vectorused was pET-29a(+), available from Novagen (Madison, Wis.). The plasmidsequence was denoted as pET-ANXA4-2X and was 7221 bp (SEQ ID NO: 16).pET-ANXA 4-2X contains an open reading frame from nucleotide number 5076to 7049 (including 3 stop codons). The first copy of Annexin IV spansnucleotides 5076-6038 of SEQ ID NO: 16, a first swivel linker spansnucleotides 6039-6044 of SEQ ID NO: 16, the PreScission proteaserecognition site spans nucleotides 6045-6068 of SEQ ID NO: 16, thesecond swivel linker spans nucleotides 6069-6074 of SEQ ID NO: 16, thesecond copy of annexin IV spans nucleotides 6081-7043 of SEQ ID NO: 16,and a kanamycin resistance gene spans nucleotides 1375-560 of SEQ ID NO:16. The sequence from nucleotide number 5076 to 7049 is furtherrepresented herein as SEQ ID NO: 17. Translation of SEQ ID NO: 17results in the annexin IV homodimer polypeptide having the followingamino acid sequence:

(SEQ ID NO: 19) MAMATKGGTVKAASGFNAMEDAQTLRKAMKGLGTDEDAIISVLAYRNTAQRQEIRTAYKSTIGRDLIDDLKSELSGNFEQVIVGMMTPTVLYDVQELRRAMKGAGTDEGCLIEILASRTPEEIRRISQTYQQQYGR R LEDDIRSDTSFMFQRVLVSLSAGGRDEGNYLDDALVRQDAQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHVFDEYKRISQKDIEQSIKSETSGSFEDALLAIVKCMRNKSAYFAEKLYKSMKGLGTDDNTLIRVMVSRAEIDMLDIRAHFKRLYGKSLYSF IKGDTSGDYRKVLLVLCGGDDGS levlfqgp SG KLAMATKGGTVKAASGFNAMEDAQTLRKAMKGLGTDEDAIISVLAYRNTAQRQEIRTAYKSTIGRDLIDDLKSELSGNFEQVIVGMMTPTVLYDVQELRRAMKGAGTDEGCLIEILA SRTPEEIRRISQTYQQQYGRR LEDDIRSDTSFMFQRVLVSLSAGGRDEGNYLDDALVRQDAQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHVFDEYKRISQKDIEQSIKSETSGSFEDALLAIVKCMRNKSAYFAEKLYKSMKGLGTDDNTLIRVMVSRAEIDMLDIRAHFKRLYGKSLYSFIKGDTSGDYRKVLLVL CGGDD

In the sequence above, the swivel sites are denoted by bold andunderline, the PreScission protease site is in lower case, and anintroduced restriction site is in italics. The annexin IV gene as clonedcontained a single base substitution compared to the published sequence(GenBank accession number NM_(—)001153) which changes the amino acid atposition 137 from serine to arginine. This change is noted in bold anddouble underline in the amino acid sequence of the dimer above.

D. Annexin VIII Homodimer. A homodimer of annexin VIII was preparedsimilarly to the annexin V homodimer described in Example 1B. The vectorused was pET-29a(+), available from Novagen (Madison, Wis.). The plasmidsequence was denoted as pET-ANXA8-2X and was 7257 bp (SEQ ID NO:20).pET-ANXA4-2X contains an open reading frame from nucleotide number 5076to 7085 (including 3 stop codons). The first copy of Annexin VIII spansnucleotides 5076-6056 of SEQ ID NO:20, a first swivel linker spansnucleotides 6057-6062 of SEQ ID NO:20, the PreScission proteaserecognition site spans nucleotides 6063-6086 of SEQ ID NO:20, the secondswivel linker spans nucleotides 6087-6092 of SEQ ID NO:20, the secondcopy of annexin VIII spans nucleotides 6099-7079 of SEQ ID NO:20, and akanamycin resistance gene spans nucleotides 1375-560 of SEQ ID NO:20.The sequence from nucleotide number 5076 to 7085 is further representedherein as SEQ ID NO:21. Translation of SEQ ID NO:21 results in theannexin VIII homodimer polypeptide having the following amino acidsequence:

(SEQ ID NO:23) MAWWKAWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTKRSNTQRQQIAKSFKAQFGKDLTETLKSELSGKFERLIVALMYPPYRYEAKELHDAMKGLGTKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQADTSGYLERILVCLLQGSRDDVSSFVDPALALQDAQDLYAAGEKIRGTDEMKFITILCTRSATHLLRVFEEYEKIANKSIEDSIKSETHGSLEEAMLTVVKCTQNLHSYFAERLYYAMKGAGTRDGTLIRNIVSRSEIDLNLIKCHFKKMYGKTLSSMIMEDTSGDYKNALLSLVGSDP GS levlfqgp SG KLAWWKAWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTKRSNTQRQQIAKSFKAQFGKDLTETLKSELSGKFERLIVALMYPPYRYEAKELHDAMKGLGTKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQADTSGYLERILVCLLQGSRDDVSSFVDPALALQDAQDLYAAGEKIRGTDEMKFITILCTRSATHLLRVFEEYEKIANKSIEDSIKSETHGSLEEAMLTVVKCTQNLHSYFAERLYYAMKGAGTRDGTLIRNIVSRSEIDLNLIKCHFKKMYGKTLSSMIMED TSGDYKNALLSLVGSDP

In the sequence above, the swivel sites are denoted by bold andunderline, the PreScission protease site is in lower case, and anintroduced restriction site is in italics. The annexin VIII gene ascloned contains a single base substitution compared to the publishedsequence (GenBank accession number NM_(—)001630). The result is a codonchange for tyrosine at position 92 from TAT to TAC.

Example 2 In Vitro and In Vivo Assays

In vitro assays determine the ability of modified annexin proteins tobind to activated platelets. Annexin V binds to platelets, and thisbinding is markedly increased in vitro by activation of the plateletswith thrombin (Thiagarajan and Tait, 1990; Sun et al., 1993).Preferably, the modified annexin proteins of the present invention areprepared in such a way that perform the function of annexin in that theybind to platelets and prevent protein S from binding to platelets (Sunet al., 1993). The modified annexin proteins also perform the functionof exhibiting the same anticoagulant activity in vitro that unmodifiedannexin proteins exhibit. A method for measuring the clotting time isthe activated partial thromboplastin time (Fritsma, in Hemostasis andthrombosis in the clinical laboratory (Corriveau, D. M. and Fritsma, G.A., eds) J.P. Lipincott Co., Philadelphia (1989), pp. 92-124,incorporated herein by reference).

In vivo assays determine the antithrombotic activity of annexinproteins. Annexin V has been shown to decrease venous thrombosis inducedby a laser or photochemically in rats (Römisch et al., 1991). Themaximal anticoagulant effect was observed between 15 and 30 minutesafter intravenous administration of annexin V, as determinedfunctionally by thromboelastography. The modified annexin proteins ofthe present invention preferably show more prolonged activity in such amodel than unmodified annexin. Annexin V was also found to decreasefibrin accretion in a rabbit model of jugular vein thrombosis (VanRyn-McKenna et al., 1993). Air injection was used to remove theendothelium, and annexin V was shown to bind to the treated vein but notto the control contralateral vein. Decreased fibrin accumulation in theinjured vein was not associated with systemic anticoagulation. Heparindid not inhibit fibrin accumulation in the injured vein. The modifiedannexin proteins of the present invention preferably perform thefunction of annexin in this model of venous thrombosis. A rabbit modelof arterial thrombosis was used by Thiagarajan and Benedict, 1997. Apartially occlusive thrombus was formed in the left carotid artery byapplication of an electric current. Annexin V infusion stronglyinhibited thrombosis as manifested by measurements of blood flow,thrombus weight, labeled fibrin deposition and labeled plateletaccumulation. Recently, a mouse model of photochemically-inducedthrombus in cremaster muscles was introduced (Vollmar et al. Thromb.Haemost. 85:160-164 (2001), incorporated herein by reference). Usingthis technique, thrombosis can be induced in any desired artery or vein.The modified annexin proteins of the present invention preferablyperform the function of annexin in such models, even when administeredby bolus injection.

Example 3

The anticoagulant ability of human recombinant annexin V and pegylatedhuman recombinant annexin V were compared in vitro.

Annexin V production. The polymerase chain reaction was used to amplifythe cDNA from the initiator methionine to the stop codon with specificoligonucleotide primers from a human placental cDNA library. The forwardprimer was 5′-ACCTGAGTAGTCGCCATGGCACAGGTTCTC-3′ (SEQ ID NO:7) and thereverse primer was 5′-CCCGAATTCACGTTAGTCATCTTCTCCACAGAGCAG-3′ (SEQ IDNO:8). The amplified 1.1-kb fragment was digested with Nco I and Eco RIand ligated into the prokaryotic expression vector pTRC 99A. Theligation product was used to transform competent Escherichia coli strainJM 105 and sequenced.

Recombinant annexin V was isolated from the bacterial lysates asdescribed by Berger et al., 1993, with some modification. An overnightculture of E. coli JM 105 transformed with pTRC 99A-annexin V wasexpanded 50-fold in fresh Luria-Bertrani medium containing 100 mg/Lampicillin. After 2 hours, isopropyl β-D-thiogalactopyranoside was addedto a final concentration of 1 mmol/L. After 16 hours of induction, thebacteria were pelleted at 3500 g for 15 minutes at 4° C. The bacterialpellet was suspended in TBS, pH 7.5, containing 1 mmol/L PMSF, 5 mmol/LEDTA, and 6 mol/L urea. The bacterial suspension was sonicated with anultrasonic probe at a setting of 6 on ice for 3 minutes. The lysate wascentrifuged at 10,000 g for 15 minutes, and the supernatant was dialyzedtwice against 50 vol TBS containing 1 mmol/L EDTA and once against 50vol TBS.

Multilamellar liposomes were prepared by dissolving phosphatidylserine,lyophilized bovine brain extract, cholesterol, and dicetylphosphate inchloroform in a molar ratio of 10:15:1 and dried in a stream of nitrogenin a conical flask. TBS (5mL) was added to the flask and agitatedvigorously in a vortex mixer for 1 minute. The liposomes were washed bycentrifugation at 3500 g for 15 minutes, then incubated with thebacterial extract, and calcium chloride was added to a finalconcentration of 5 mmol/L. After 15 minutes of incubation at 37° C., theliposomes were sedimented by centrifugation at 10,000 g for 10 minutes,and the bound annexin V was eluted with 10 mmol/L EDTA. The elutedannexin V was concentrated by Amicon ultrafiltration and loaded onto aSephacryl S 200 column. The annexin V was recovered in the includedvolume, whereas most of the liposomes were in the void volume. Fractionscontaining annexin V were pooled and dialyzed in 10 mmol/L Tris and 2mmol/L EDTA, pH 8.1, loaded onto an anion exchange column, and elutedwith a linear gradient of 0 to 200 mmol/L NaCl in the same buffer. Thepurified preparation showed a single band in SDS-PAGE under reducingconditions.

The annexin V produced as above was pegylated using the method ofHermanson, 1996, as described above.

Anti-coagulation assays. Prolongation of the clotting time (activatedpartial thromboplastin time) induced by annexin V and pegylated annexinV were compared. Activated partial thromboplastin times were assayedwith citrated normal pooled plasma as described in Fritsma, 1989. Usingdifferent concentrations of annexin V and pegylated annexin V, producedas described above, dose-response curves for prolongation of clottingtimes were obtained. Results are shown in FIG. 6, a plot of clottingtime versus annexin V and pegylated annexin V dose. As shown in thefigure, the anticoagulant potency of the recombinant human annexin V andthe pegylated recombinant human annexin V are substantially equivalent.The small difference observed is attributable to the change in molecularweight after pegylation. This experiment validates the assertion madeherein that pegylation of annexin V can be achieved withoutsignificantly reducing its antithrombotic effects.

Example 4

The affinities of recombinant annexin V (AV) and recombinant annexin Vhomodimer (DAV, Diannexin) for PS on the surface of cells were compared.To produce cells with PS exposed on their surfaces, human peripheral redblood cells (RBCs) were treated with a Ca²⁺ ionophore (A23187). Thephospholipid translocase (flipase), which moves PS to the inner leafletof the plasma membrane bilayer, was inactivated by treatment withN-ethyl maleimide (NEM), which binds covalently to free sulfhydrylgroups. Raising intracellular Ca²⁺ activates the scramblase enzyme, thusincreasing the amount of PS in the outer leaflet of the plasma membranebilayer.

Washed human RBCs were resuspended at 30% hematocrit in K-buffer (80 mMKCI, 7 mM NACI, 10 mM HEPES, pH 7.4). They were incubated for 30 minutesat 37° C. in the presence of 10 mM NEM to inhibit the flipase. TheNEM-treated cells were washed and suspended at 16% hematocrit in thesame buffer with added 2 mM CaCl₂. The scramblase enzyme was activatedby incubation for 30 minutes at 37° C. with A23187 (final concentration4 μM). As a result of this procedure, more than 95% of the RBCs had PSdemonstrable on their surface by flow cytometry.

Recombinant AV and DAV were biotinylated using the FluReporterprotein-labeling kit (Molecular Probes, Eugene Oreg.). Biotin-AV andbiotin-DAV conjugates were visualized with R-phycoerythrin-conjugatedstreptavidin (PE-SA) at a final concentration of 2 μg/ml. Flow cytometrywas performed on a Becton Dickinson FACScaliber and data were analyzedwith Cell Quest software (Becton Dickinson, San Jose Calif.).

No binding of AV or DAV was detectable when normal RBCs were used.However, both AV and DAV were bound to at least 95% of RBCs exposing PS.RBCs exposing PS were incubated with various amounts of AV and DAV,either (a) separately or (b) mixed in a 1:1 molar ratio, before additionof PE-SA and flow cytometry. In such mixtures, either AV or DAV wasbiotinylated and the amount of each protein bound was assayed asdescribed above. The experiments were controlled for higher biotinlabeling in DAV than AV.

Representative results are shown in FIG. 2. In this set of experiments,RBCs exposing PS were incubated with (a) 0.2 μg of biotinylated DAV(FIG. 2A); (b) 0.2 μg of biotinylated DAV (FIG. 2B); (c) 0.2 μg ofbiotinylated AV and 0.2 μg nonbiotinylated DAV; and (d) 0.2 μg ofbiotinylated DAV and 0.2 pg nonbiotinylated AV (FIG. 2D). Comparing FIG.2B and FIG. 2D shows that the presence of 0.2 μg of nonbiotinylated AVhad no effect on the binding of biotinylated DAV. However, comparingFIG. 2A and FIG. 2C shows that the presence of 0.2 μg of nonbiotinylatedDAV strongly reduced the amount of biotinylated AV bound to PS-exposingcells. These results indicate that DAV and AV compete for the samePS-binding sites on RBCs, but with different affinities; DAV binds to PSthat is exposed on the surface of cells with a higher affinity than doesAV.

Example 5

A cell-binding assay was established using known amounts of annexin Vmonomer (AV) and dimer (DAV) added to mouse serum. RBCs withexternalized PS, as described above, were incubated with serumcontaining dilutions of AV and DAV. After washing, addition of labeledstreptavidin and washing again, AV and DAV bound to the RBCs wereassayed by flow cytometry. No binding was detectable when RBCs withoutexternalized PS were used. Concentrations of AV and DAV in mouse serum,assayed by cell binding, were highly correlated with those determined byindependent ELISA assays. Hence, AV and DAV in mouse plasma are notbound to other plasma proteins in a way that impairs their capacity tointeract with externalized PS on cell surfaces. These observationsvalidated the application of the cell-binding assay to compare thesurvival of AV and DAV in the circulation.

Mice were injected intravenously with AV and DAV, and peripheral bloodsamples were recovered at several times thereafter. Different mice wereused for each time point. Representative results are shown in FIG. 3.Observations in the rabbit (Thiagarajan and Benedict, Circulation 96:2339, 1977), cynomolgus monkey, (Römisch et al., Thrombosis Res. 61: 93,1991) and humans (Kemerink et al., J. Nucl. Med. 44: 947, 2003) showthat AV has a short half-life in the circulation (7 to 24 minutes,respectively), with a major loss into the kidney. Consistent with thesereports, 20 minutes after injection of AV into the mouse, virtually nonewas detectable in the peripheral blood (FIG. 3B). However, even 120minutes after intravenous injection of DAV into mice, substantialamounts of the protein were detectable in the circulation (FIG. 3E).Thus dimerization of annexin V increases its survival in the circulationand hence the duration of its therapeutic efficacy.

Example 6

The inhibitory effects of annexin V (AV) and the annexin V homodimer(DAV) on the activity of human sPLA₂ (Cayman, Ann Arbor Mich.) werecompared. PS externalized on RBCs treated with NEM and A23 187, asdescribed above, was used as the substrate. In control cells, AV and DAVwere found to bind to PS-exposing RBCs as demonstrable by flowcytometry. Incubation of the PS-exposing cells with sPLA₂ removes PS, sothat the cells no longer bind annexin. If the PS-exposing cells aretreated with AV or DAV before incubation with PLA₂, the PS is notremoved. The cells can be exposed to a Ca²⁺-chelating agent, whichdissociates AV or DAV from PS, and subsequent binding of labeled AVreveals the residual PS on cell surfaces. Titration of AV and DAV insuch assays shows that both are potent inhibitors of the activity ofsPLA₂ on cell-surface PS.

The inhibition of phospholipase is also demonstrable by another method.Activity of sPLA₂ releases lysophosphatidylcholine (LPS), which ishemolytic. It is therefore possible to compare the inhibitory effects ofAV and DAV on PLA₂ in a hemolytic assay. As shown in FIG. 4, both AV andDAV inhibit the action of PLA₂, with DAV being somewhat moreefficacious. Hemolysis induced after 60 minutes incubation with pPLA₂was strongly reduced in the presence of DAV or AV compared to theirabsence. From these results it can be concluded that the homodimer ofannexin V is a potent inhibitor of secretory PLA₂. It should thereforedecrease the formation of mediators such as thromboxane A2, as well aslysophosphatidylcholine and lysophosphatidic acid, which are believed tocontribute to the pathogenesis of reperfusion injury (Hashizume et al.Jpn. Heart J., 38: 11, 1997; Okuza et al., J. Physiol., 285: F565,2003).

Example 7

A mouse liver model of warm ischemia-reperfusion injury was used toascertain whether modified annexins protect against reperfusion injury(RI), compare the activity of annexin V with modified annexins, anddetermine the duration of activity of modified annexins. The model hasbeen described by Teoh et al. (Hepatology 36:94, 2002). Female C57BL6mice weighing 18 to 25 g were used. Under ketamine/xylazine anesthesia,the blood supply to the left lateral and median lobes of the liver wasoccluded with an atraumatic microvascular clamp for 90 minutes.Reperfusion was then established by removal of the vascular clamp. Theanimals were allowed to recover, and 24 hours later they were killed byexsanguination. Liver damage was assessed by measurement of serumalanine aminotransferase (ALT) activity and histological examination. Acontrol group was subjected to anesthesia and sham laparotomy. To assaythe activity of annexin V and modified annexins, groups of 4 mice wereused. Each of the mice in the first group was injected intravenouslywith 25 micrograms of annexin V (AV), each of the second group received25 micrograms of annexin homodimer (DAV), and each of the third groupreceived 2.5 micrograms of annexin V coupled to polyethylene glycol(PEG-AV, 57 kDa). Controls received saline or the HEPES buffer in whichthe annexins were stored. In the first set of experiments, the annexinswere administered minutes before clamping branches of the hepaticartery. In the second set of experiments, annexins and HEPES wereadministered 6 hours before initiating ischemia. Representativeexperimental results are summarized in FIG. 5.

In animals receiving annexin V (AV) just before ischemia, slightprotection was observed. By contrast, animals receiving the annexindimer (DAV) or PEG-AV, either just before or 6 hours before ischemia,showed dramatic protection against RI. Histological studies confirmedthat there was little or no hepatocellular necrosis in these groups. Theresults show that the modified annexins (DAV and PEG-AV) aresignificantly more protective against ischemia reperfusion injury in theliver than is AV. Furthermore, the modified annexins (DAV and PEG-AV)retain their capacity to attenuate RI for at least 6 hours.

In sham-operated animals, levels of ALT in the circulation were verylow. In animals receiving saline just before ischemia, or HEPES 6 hoursbefore ischemia, levels of ALT were very high, and histology confirmedthat there was severe hepatocellular necrosis. HEPES administered justbefore ischemia was found to have protective activity against RI.

Example 8

Thrombosis Study

Six groups of eight rats each were used. The rats for this study weremale Wistar rats, weighing about 300 grams (Charles River Nederland,Maastricht, the Netherlands). Animals were housed in macrolon cages, andgiven standard rodent food pellets and acidified tap water ad lib.Experiments conformed to the rules and regulations set forward by theNetherlands Law on Animal Experiments. Rats were anaesthetized with FFM(Fentanyl/Fluanison/Midazolam), and placed on a heating pad. A cannulawas inserted into the femoral vein and filled with saline. The vena cavainferior was isolated, and side branches were closed by ligation orcauterization. A loose ligature was applied around the caval vein belowthe left renal vein. A second loose ligature was applied 1.5 cm upstreamfrom the first one, above the bifurcation. The test (or control)compound was given intravenously via the femoral vein cannula, and thecannula was then flushed with saline.

Test or control compounds include phosphate-buffered saline 1.0 ml/kgbodyweight (10 min); Phosphate-buffered saline 1.0 ml/kg bodyweight (12hrs); Diannexin 0.04 mg/kg body weight; Diannexin 0.2 mg/kg body weight;Diannexin 1.0 mg/kg body weight (10 min); Diannexin 1.0 mg/kg bodyweight (12 hrs); Fragmin 20 aXa U/kg body weight. Ten minutes later (orin two groups: 12 hrs later), recombinant human thromboplastin (0.15mL/kg) was rapidly injected into the venous cannula, the cannula wasflushed with saline, and exactly ten seconds later the downstreamligature near the renal vein was closed. After nine minutes, a citratedvenous blood sample was obtained and put on ice.

One minute later (at ten minutes) the upstream ligature near thebifurcation was closed and the thrombus that had formed in the segmentwas recovered. The thrombus was briefly washed in saline, blotted, andits wet weight was determined. Citrated plasma was prepared bycentrifugation for 15 min at 2000 g at 4° C., and stored at −60° C. foranalysis. In the two groups in which thrombus induction took place at 12hrs after compound injection, a different i.v. injection procedure wasused. Rats were anaesthetized with s.c. DDF (Domitor/Dormicum/Fentanyl)and injected via the vein of the penis. Rats were then s.c. given anantidote (Anexate/Antisedan/Naloxon) and kept overnight in their cage.

After insertion of a femoral vein cannula, rats were intravenouslyinjected with Diannexin or Fragmin. At 10 minutes after the intravenousinjection of compound (in two groups: at 12 hrs after injection),diluted thromboplastin was injected i.v., and ten seconds later the venacava inferior ligated. At nine minutes after ligation, blood wascollected and citrated plasma was prepared. At ten minutes afterligation, the thrombosed segment was ligated, and the thrombus wasrecovered and weighed. aPTT (sec) was also measured. At 12 hrs afterinjection of Diannexin decreased the thrombus weight in a dose-dependentmanner. (FIG. 7). At 1 mg/kg, suppression of thrombosis was nearlycomplete, and not significantly different from that produced by thereference anti-thrombotic drug, the low molecular weight heparindesignated Fragmin.

TABLE 1 Effect of Treatment on Thrombus Wet Weight (mg) in the 10-minThrombosis study. Diannexin Diannexin Diannexin Fragmin 20 Saline 1mg/kg 0.2 mg/kg 0.04 mg/kg aXa U/kg 21.0 1.8 0.0 15.5 0.5 43.8 0.0 4.319.6 1.5 26.6 3.2 2.1 220 4.6 44.5 0.5 6.0 7.5 00 17.6 3.5 3.1 10.5 4.324.0 2.7 2.8 15.6 30 10.6 4.3 5.2 16.6 0.0 17.8 0.5 4.7 15.3 0.0 mean25.7 2.1 3.5 15.3 1.7 sd 12.3 1.6 1.9 4.6 20 By parametric ANOVA; F =24.48; p < 0.00001 All groups < saline controls (p < 0.01) By parametricANOVA of the three Diannexin groups: F = 4600, p < 0.0001 1 mg = 0.2 mg< 0.04 mg; p < 0.001

Treatment had a significant effect on thrombus weight. Both Fragmin (20.aXa U/kg) and Diannexin (0.04, 0.2 and 1.0 mg/kg) significantly reducedthrombus weight (p<0.000 1), see Table 1. For Diannexin, the effect wasdose-dependent. The APTT values are shown in Table 2.

TABLE 2 Effect of Treatment on the APTT (seconds) in the 10-MinuteThrombosis Study Diannexin Diannexin Diannexin Fragmin 20 Saline 1 mg/kg0.2 mg/kg 0.04 mg/kg aXa U/kg 20.7 26.1 17.6 20.7 n.a. 20.0 22.0 20.823.5 27.1 17.6 19.0 20.7 22.0 37.9 21.6 16.5 20.2 21.7 19.5 17.5 21.521.3 24.9 24.2 14.7 23.0 23.0 21.5 24.4 20.2 22.5 19.0 19.9 29.7 18.719.3 20.4 19.4 25.0 mean 18.9 21.2 20.4 21.7 26.8 sd 2.2 2.9 1.6 1.8 5.8By parametric ANOVA; F = 6.66; p = 0.0005 Fragmin group > all othergroups (p < 0.05) Saline and Diannexin groups not significantlydifferent

Fragmin increased the APPT significantly, compared to all other groups.The APTT was slightly, though significantly increased only in theFragmin group. The Diannexin groups did not differ from the salinecontrol group.

In the second thrombosis study, in which rats were treated at 12 hrsbefore the induction of thrombus formation, no significant differencebetween the saline-injected control group and the Diannexin-treatedgroup was found (Table 3).

TABLE 3 Effect of Treatment on Thrombus Wet Weight (mg) in the 12-hrThrombosis study. Diannexin Saline 1 mg/kg 16.1 22 21.2 9.5 17.1 13.523.2 29.0 15.3 22.1 19.2 18.3 15.6 22.3 20.8 37.9 mean 18.6 21.8 sd 38.8 *mean time to thrombus induction: 13.6 hrs no significant differenceby t-test

Thrombus weights in the saline group were also not significantlydifferent from thrombus weights in the saline control group of the10-min thrombosis study (25.7±12.3 mg, see Table 1). APTT values werenot different (not shown).

In summary, the observations show that Diannexin has potentantithrombotic activity in the dose range 0.2 to 1 mg/kg. This effect isno longer demonstrable 12 hours after injection. In the unlikely eventthat Diannexin produces hemorrhage or any other adverse effect, thepatient should soon recover

Example 9

Bleeding study: Three groups were studied. Groups of eight rats, asdescribed in Example 8, were used. Rats were anaesthetized withisoflurane, intubated and ventilated, and placed on a heating pad. Acannula was inserted into the femoral vein, and filled with saline. Testor control compounds were i.v. injected via the cannula, and the cannulawas then flushed with saline. Test or control compound werephosphate-buffered saline 1.0 ml/kg bodyweight; Diannexin 5.0 mg/kg bodyweight; Fragmin 140 aXa U/kg body weight. At 10 min after injection oftest compound, the rat tail was put in a horizontal position, and thentransected at a defined fixed distance from the tail by scissors.Subsequently, bleeding from the tail was determined by gentlyblotting-off all blood protruding from the tail by filter paper. Thetime when bleeding stopped was determined. Any was noted. The experimentwas terminated at 30 mm after tail transection. Just prior to the end ofthe experiment, a citrated blood sample was obtained from the cannula.Citrated plasma was prepared by centrifugation for 15 min at 2000 g at−40° C., and stored at −60° C. for analysis. The filter papers wereextracted in 20 ml of 10 mM phosphate buffer (pH=7.8), containing 0.05%Triton X-100®. The amount of blood lost was determined by measuring thehemoglobin content of the phosphate buffer (potassium cyanide 1potassium fefficyanide procedure of Drabkin). Body weight (Table 3) didnot differ between groups by parametric ANOVA. Treatment by eitherDiannexin (mg/kg) 140 U/kg approximately doubled bleeding time (FIG. 9,Table 3), although these effects were only borderline significant(nonparametric; ANOVA; KW=5.72, p=0.057). Blood loss (FIG. 10, Table 4)was slightly increased in the Diannexin group, and immediately doubledin the Fragmin group, compared to the control group.

TABLE 4 Bleeding times and Blood Loss in the Tail Bleeding Study primarySecondary bleeding bleeding blood rat # time (min) (min) loss (mL)SALINE GROUP 1 2.5 # 0.049 2 30.0 # 0.400 3 17.67 # 0.58 4 110 5.5 0.0355 30.0 # 0.384 6 10 # 0.001 7 7.5 2.0 0.009 8 8.67 # 0.034 mean 13.50.19 sd 11.4 0.23 median 9.8 0.042 DIANNEXIN GROUP 1 30.0 # 0.257 216.16 # 0.016 3 300 # 0.022 4 180 10.0 0.098 5 30.0 # 0.263 6 17.0 10.01.868 7 30.0 # 0.107 8 30.0 # 0.037 mean 25.1 # 0.33 sd 6.7 0.63 median30 0.104 FRAGMIN GROUP 1 12.0 12.0 0.034 2 9.0 8.67 0.069 3 30.0 # 0.2634 30.0 # 0.093 5 15.0 # nd 6 30.0 # 1.846 7 30.0 # 1.520 8 30.0 # 0.213mean 23.3 0.58 sd 9.5 0.77 median 30 0.213

These differences were, however, not significant (non-parametric ANOVA,p=0.490). The APTT values are shown in Table 5 and in FIG. 11.

TABLE 5 Effect of Treatment on the APTT (seconds) in the Tail BleedingStudy. Diannexin Fragmin 20 Saline 5 mg/kg aXa U/kg 24.3 26.3 46.6 17.827.0 32.1 17.3 24.1 62.9 16.5 25.5 69.8 19.9 27.7 69.1 20.3 25.1 52.421.4 21.0 45.7 21.9 23.2 56.5 mean 19.9 25.2 54.4 sd 2.6 2.2 12.9

Fragmin approximately doubled the APTT, while the APTT in the Diannexingroup did not differ from the saline control group (FIG. 11).

Blood loss and the aPTT were approximately twice as large in the Fragmingroup as in the Diannexin group in the tail bleeding study. At 5.0 mg/kgi.v. Diannexin induced bleeding from a transected rat tail, though lessblood was lost than after injection of 140 aXa U/kg of Fragmin.

Example 10

Clearance study. Rats were injected with radiolabeled Diannexin, bloodsamples were obtained at 5, 10, 15, 20, 30, 45, and 60 min and 2, 3, 4,8, 16 and 24 hrs, and blood radioactivity was determined to construct ablood disappearance curve. (FIG. 12) Disappearance of Diannexin fromblood could be described by a two-compartment model, with about 75-80%disappearing in the α-phase (t/2 about 10 min), and 15-20% in theβ-phase (t/2 about 400 min). Clearance could be described by atwo-compartment model, with half-lives of 9-14 min and 6-7 hrs,respectively. Two experiments were performed, each with three maleWistar rats (300 gram). Diannexin was labelled with ¹²⁵I by the methodof Macfarlane, and the labeled protein was separated from free SephadexG-50. After injection of NaI (5mg/kg) to prevent thyroid uptake oflabel, about 8×10⁶ cpm (50 μL of protein solution diluted to 0.5 mL withsaline) were injected via a femoral vein catheter (rats 1 and 2) or viathe vein of the penis (rat 3). At specified times thereafter (see Tablebelow), blood samples (150 μL) were obtained from a tail vein and 100 μLwas counted. After the last blood sample, rats were sacrificed byNembutal i.v., and (pieces of) liver, lung, heart, spleen and kidneyswere collected for counting.

The β-phase parameters were calculated from the data collected between45 min and 24 hrs. The α-phase parameters were then calculated from thedata between 5 and 45 min by the subtraction method. The bloodradioactivity curves were analysed by a two-compartment model, using thesubtraction method. The linear correlation coefficients for the α- andthe β-phase were −0.99 and −0.99 in experiment 1, and −0.95 and −0.96 inexperiment 2. The clearance parameters are shown in Table 6.

TABLE 6 Diannexin clearance parameters. Experiment 1 Experiment 2 t/2alpha phase  9.2 min 14.1 min t/2 beta phase 385 min  433 min % in alphaphase 85% 79% % in beta phase 15% 21% Isotype recovery in blood (%) 89%52%

FIGS. 15 and 16 show the clearance curves with the alpha- andbeta-phases superimposed. In Table 7 are shown the cpm recovered inlung, heart, liver spleen and kidneys (after digestion of the tissues).Of note is the high number of counts in the lung at 2 hrs afterDiannexin injection.

TABLE 7 Radioactivity Recovered in Selected Tissues at 2, 8 and 24 hoursafter injection of ¹²⁵I-Diannexin. cpm/tissue % of total counts at 2 hrsat 8 hrs at 24 hrs at 2 hrs at 8 hrs at 24 hrs Exp 1 lung 166740 416224228 28 16 5 spleen 82425 15211 4074 14 6 5 heart 22582 11144 1610 4 4 2liver 181832 85359 19730 30 33 24 kidneys 151858 108241 53046 25 41 64sum 605437 261577 82688 100 100 100 % of 2 hrs 100 43 14 Exp 2 lung242130 12495 4025 47 8 6 spleen 55377 11466 5019 11 7 7 heart 14966 81271645 3 5 2 liver 37628 7152 1642 7 5 2 kidneys 168560 114030 60774 32 7483 sum 518661 153270 73105 100 100 100 % of 2 hrs 100 30 14

Example 11

Studies were undertaken to confirm the pathogenesis ofischemia-reperfusion injury (IRI) and mode of action of Diannexin.According to the hypothesis of the pathogenesis of ischemia-reperfusioninjury which is part of the present invention, during ischemia,phosphatidylserine (PS) becomes accessible on the luminal surface ofendothelial cells (EC) in the hepatic microvasculature. During thereperfusion phase leukocytes and platelets become attached to PS on thesurface of EC the surface of EC and reduce blood flow in the hepaticmicrocirculation. Diannexin binds to PS on the surface of EC anddecreases the attachment of leukocytes and platelets to them. By thismechanism Diannexin maintains blood flow in the hepatic microcirculationand thereby attenuates ischemia-reperfusion injury.

This hypothesis was tested by observing the microcirculation in themouse liver in vivo using published methods (McCuskey et al., Hepatology40: 386,2004). As described in example 7, 90 minutes of ischemia wasfollowed by various times of reperfusion. FIGS. 12A and 12B show thatduring reperfusion many leukocytes become attached to EC in both theperiportal and centrilobular areas (IR). Diannexin (1 mg/kg) IV) reducessuch attachment in a statistically significant manner (IR+D). FIGS. 13Aand 13B show that this is also true of the adherence of platelets to ECduring reperfusion. As predicted, EC damage (reflected by swelling) isprominent during reperfusion and is significantly decreased by Diannexin(FIG. 14A and 14B). Our hypothesis of the mode of action of Diannexin inattenuating ischemia-reperfusion injury is therefore confirmed. As shownin FIGS. 15A and 15B, Diannexin does not influence the phagocyticactivity of Kupffer cells in either location. Hence, Diannexin has noeffect on this defense mechanism against pathogenic organisms. Thisfinding supports other evidence that Diannexin does not have adverseeffects.

Example 12

The efficacy of Diannexin in protection of organs of cold ischemia-warmreperfusion injury was evaluated in a rat liver transplantation model(Sawitzki, B. et al. Human Gene Therapy 13: 1495, 2002). Livers wererecovered from adult male Sprague-Dawley rats, perfused with Universityof Wisconsin solution, kept at 4° C. for 24 hrs and transplantedorthotopically into syngeneic recipients. Under these conditions 60% ofuntreated recipients died within 48 hours of transplantation, aspreviously observed in similar experiments. Another 10 recipients ofliver grafts were given Diannexin (0.2 mg/kg intravenously) 10 minutesand 24 hrs after transplantation. All these animals survived for morethan 14 days, which on the basis of previous experience implies survivalunlimited by the operation.

As shown in table 8, levels of the liver enzyme alanine aminotransferase(ALT) in the circulation of untreated recipients at 6 hrs and 24 hrsafter transplantation were significantly higher than inDiannexin-treated recipients. Diannexin-mediated cytoprotection wasconfirmed by histological examination of the livers in transplantrecipients. By 7 days after transplantation ALT levels were back to thenormal range in all recipients.

In second group of 10 recipients Diannexin was used in a different way.Rat livers were obtained from Sprague-Dawley donors and perfused ex vivowith University of Wisconsin Solution containing Diannexin (0.2mg/liter) twice, before 24 hr of 4° cold storage and just beforeorthotopic transplantation. No Diannexin was given post-transplant tothese recipients, all of which survived >14 days. Again ALT levels at 6and 24 hrs were significantly lower than in untreated animals andhistological examination showed a substantial difference between thewell preserved livers in Diannexin-treated and the partially necroticlivers in control graft recipients.

These observations show that Diannexin markedly attenuates IRI in a coldischemia-warm reperfusion rat liver model which is similar to thesituation in human liver transplantation. Diannexin is equallyefficacious when included in the solution used to perfuse the liver exvivo when administered to recipients of liver grafts shortly aftertransplantation.

TABLE 8 Serum ALT levels (IU/L) in rat liver graft recipients (mean ±SD) Untreated controls Diannexin treated P value 6 hrs 1345 ± 530 267 ±110 <0.001 1 day 4031 ± 383 620 ± 428 <0.001 7 days  99 ± 31 72 ± 8 >0.5

Example 12

As an experimental model of human steatotic livers mice were madesteatotic by feeding them a diet containing 20% fat for 8 weeks. Groupsof 5 female C57BL6 mice weighing 18 to 25 g were subjected to 90 minutesischemia and 24 hours reperfusion. One group of recipients received 1mg/kg Diannexin just before the commencement of reperfusion. As shown bythe liver enzyme levels in FIG. 16, Diannexin provided highlysignificant protection against IRI.

Example 13

This experiment was undertaken to ascertain whether Diannexin canprotect the liver from IRI when administration of the protein is delayeduntil after the commencement of reperfusion. Our standard protocol forthe mouse liver warm IRI was used: adult female C57BL6 mice, 90 minutesischemia and 24 hrs reperfusion. Endpoints were serum ALT levels andliver pathology at 24 hours. Diannexin (1 mg/kg) was administered 10minutes and 60 minutes after commencement of reperfusion. As shown intable 9, both of these procedures significantly decreased ALT levels,and protective effects were confirmed by liver histology. Theseobservations show that Diannexin administration can be delayed until atleast 1 hour after the initiation of reperfusion, implying thatendothelial changes during the first hour are reversible. The findingsalso show that administration of Diannexin a few minutes afterre-establishing the circulation in recipients of transplanted organsshould attenuate IRI.

TABLE 9 Effect of Diannexin (1 mg/kg) Administration during ReperfusionTime after commencement Serum ALT of reperfusion mean ± s.d. Probability 0 (untreated control)  840 ± 306 10 minutes 153 ± 83 p < 0.05 60minutes 255 ± 27 p < 0.05

1. A composition comprising an isolated annexin protein selected fromthe group consisting of: (a) an annexin homodimer at least 95% identicalto a protein selected from the group consisting of SEQ ID NO: 6, SEQ IDNO: 27, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 3-linker-SEQ ID NO: 3,SEQ ID NO: 12-linker-SEQ ID NO: 12, and SEQ ID NO: 15-linker-SEQ ID NO:15, wherein the linker is at least one glycine-serine sequence; (b) anannexin heterodimer at least 95% identical to a protein selected fromthe group consisting of SEQ ID NO: 3-linker-SEQ ID NO: 12, SEQ ID NO:3-linker-SEQ ID NO: 15, SEQ ID NO: 12-linker-SEQ ID NO: 15, SEQ ID NO:12-linker-SEQ ID NO: 3, SEQ ID NO: 15-linker-SEQ ID NO: 3, and SEQ IDNO: 15-linker-SEQ ID NO:12, wherein the linker is at least oneglycine-serine sequence; and (c) an annexin protein coupled to one ormore polyethylene glycol (PEG) chains wherein the annexin protein is atleast 95% identical to a protein selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 15, wherein said isolatedannexin protein has phosphatidylserine binding activity.
 2. Thecomposition of claim 1, wherein each polyethylene glycol chain has amolecular weight of at least 10 kDa.
 3. The composition of claim 1,wherein each polyethylene glycol chain has a molecular weight of atleast 30 kDa.
 4. The composition of claim 1, wherein the isolatedannexin protein is coupled to two or more polyethylene glycol chains,and wherein the isolated annexin protein is selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:12 and SEQ ID NO:15.
 5. Thecomposition of claim 4, wherein the isolated annexin protein is SEQ IDNO: 12-PEG.
 6. The composition of claim 4, wherein the isolated annexinprotein is SEQ ID NO: 3-PEG.
 7. The composition of claim 4, wherein theisolated annexin protein is SEQ ID NO: 15-PEG.
 8. A pharmaceuticalcomposition comprising a therapeutically effective amount of an isolatedannexin protein and an excipient, said isolated annexin protein isselected from the group consistinci of: (a) an annexin homodimer atleast 95% identical to a protein selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 27, SEQ ID NO: 19 SEQ ID NO: 23, SEQ ID NO:3-linker-SEQ ID NO: 3, SEQ ID NO: 12 -linker-SEQ ID NO: 12, and SEQ IDNO: 15-linker-SEQ ID NO: 15, wherein the linker is at least oneglycine-serine sequence; (b) an annexin heterodimer at least 95%identical to a protein selected from the group consisting of SEQ ID NO:3 -linker-SEQ ID NO: 12, SEQ ID NO: 3 -linker-SEQ ID NO: 15, SEQ ID NO:12 -linker-SEQ ID NO: 15, SEQ ID NO: 12 -linker -SEQ ID NO: 3, SEQ IDNO: 15-linker-SEQ ID NO: 3, and SEQ ID NO: 15-linker-SEQ ID NO: 12,wherein the linker is at least one glycine-serine sequence; and (c) anannexin protein coupled to one or more polyethylene glycol chainswherein the annexin protein is at least 95% identical to a proteinselected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, andSEQ ID NO: 15, wherein the isolated annexin protein has a prolongedhalf-life in a vascular compartment of a subject in need of saidcomposition compared to a wild-type annexin and the isolated annexinprotein has an increased affinity for phosphatidylserine.
 9. Thepharmaceutical composition of claim 8, wherein the isolated annexinprotein is an annexin protein at least 95% identical to SEQ ID NO: 12.10. The pharmaceutical composition of claim 8, wherein the isolatedannexin protein is an annexin protein at least 95% identical to SEQ IDNO:
 3. 11. The pharmaceutical composition of claim 8, wherein theisolated annexin protein is an annexin protein at least 95% identical toSEQ ID NO:
 15. 12. The composition of claim 1, wherein said isolatedannexin protein is selected from the group consisting of SEQ ID NO: 6,SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO:23, SEQ ID NO: 3-linker-SEQ IDNO: 12, SEQ ID NO: 3-linker-SEQ ID NO: 15, SEQ ID NO: 12-linker-SEQ IDNO: 15, SEQ ID NO: 12-linker-SEQ ID NO: 3, SEQ ID NO: 15-linker-SEQ IDNO: 3, and SEQ ID NO: 15-linker-SEQ ID NO:12, wherein the linker is atleast one glycine-serine sequence.
 13. The pharmaceutical composition ofclaim 8, wherein said isolated annexin protein is selected from thegroup consisting of SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 27, SEQ IDNO:23, SEQ ID NO: 3-linker-SEQ ID NO: 12, SEQ ID NO: 3-linker-SEQ ID NO:15, SEQ ID NO: 12-linker-SEQ ID NO: 15, SEQ ID NO: 12-linker-SEQ ID NO:3, SEQ ID NO: 15-linker-SEQ ID NO: 3, and SEQ ID NO: 15-linker-SEQ IDNO:12, wherein the linker is at least one glycine- serine sequence.